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Sample records for real-time rt-pcr analysis

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

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    Bester Rachelle

    2012-09-01

    Full Text Available Abstract 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

  2. Monitoring gene expression: quantitative real-time rt-PCR.

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    Wagner, Elke M

    2013-01-01

    Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time.RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification or absolute quantification compared to standards that are run in parallel can be performed.This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addition, tips for choosing primers and performing analyses are provided to help the beginner in understanding the technique.

  3. Use of Real-time RT-PCR Analysis for mRNA Expression of Tobacco Ferritin Gene (NtFer1)

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    JIANG Tingbo; LI Fengjuan; YANG Chuanping

    2006-01-01

    To understand the use of real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for detecting the relative abundance of mRNA, the expression of a tobacco ferritin gene (NtFer1) was detected by Northern blot and real-time RT-PCR. The results indicated that both of the two methods were able to detect mRNA expression of NtFer1 clearly and similarly, namely NtFer1 expression was responsive to iron-overload, and the abundance of NtFer1 mRNA was greatly increased after iron loaded for 6 h. To compare the effect and sensitivity of two methods, results revealed that Northern blot need 30 μg of total RNA and at least 3 days for the total protocol performance, whereas real-time RT-PCR only need 2 μg of total RNA and 1.5 h. The real-time RT-PCR is rather sensitive and effective than Northern blot. Real-time RT-PCR analysis can be used to rapidly detect the relative abundance of mRNA expression instead of Northern blot analysis.

  4. Two metallothionein gene family members in buckwheat: Expression analysis in flooding stress using Real Time RT-PCR technology

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    Majić Dragana B.

    2008-01-01

    Full Text Available Metallothioneins (MTs are an extensive and diverse family of small cysteine-rich proteins with metal-binding ability that are involved in metal homeostasis and detoxification. Two cDNA clones of the MT3 type, differing in 3’ UTRs, were isolated from the developing buckwheat seed cDNA library. Following sequence analyses, expression profiles during flooding stress were monitored by Real Time RT PCR technology.

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

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

    2017-09-04

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

  6. A plastome primer set for comprehensive quantitative real time RT-PCR analysis of Zea mays: a starter primer set for other Poaceae species

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    Dunn Sade N

    2008-06-01

    Full Text Available Abstract Background Quantitative Real Time RT-PCR (q2(RTPCR is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RTPCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species. Results Primers were designed and successfully optimized for 57 of the 104 reported genes in the maize plastome plus two nuclear genes. All 59 primer pairs produced single amplicons after end-point reverse transcriptase polymerase chain reactions (RT-PCR as visualized on agarose gels and subsequently verified by q2(RTPCR. Primer pairs were divided into several categories based on the optimization requirements or the uniqueness of the target gene. An in silico test suggested the majority of the primer sets should work with other members of the Poaceae family. An in vitro test of the primer set on two unsequenced species (Panicum virgatum and Miscanthus sinensis supported this assumption by successfully producing single amplicons for each primer pair. Conclusion Due to the highly conserved chloroplast genome in plant families it is possible to utilize primer pairs designed against one genomic sequence to detect the presence and abundance of plastid genes or transcripts from genomes that have yet to be sequenced. Analysis of steady state transcription of vital system genes is a necessary requirement to comprehensively elucidate gene expression in any organism. The primer pairs reported in this paper were designed for q2(RTPCR of maize chloroplast genes but should be useful for other members of the Poaceae family. Both in silico and in vitro data are presented to support this assumption.

  7. Selection of ovine housekeeping genes for normalisation by real-time RT-PCR; analysis of PrP gene expression and genetic susceptibility to scrapie

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    Hurtado Ana

    2005-09-01

    Full Text Available Abstract Background Cellular prion protein expression is essential for the development of transmissible spongiform encephalopathies (TSEs, and in sheep, genetic susceptibility to scrapie has been associated to PrP gene polymorphisms. To test the hypothetical linkage between PrP gene expression and genetic susceptibility, PrP mRNA levels were measured by real-time RT-PCR in six ovine tissues of animals with different genotypes. Results Previous to the PrP gene expression analysis the stability of several housekeeping (HK genes was assessed in order to select the best ones for relative quantification. The normalisation of gene expression was carried out using a minimum of three HK genes in order to detect small expression differences more accurately than using a single control gene. The expression stability analysis of six HK genes showed a large tissue-associated variation reflecting the existence of tissue-specific factors. Thereby, a specific set of HK genes was required for an accurate normalisation of the PrP gene expression within each tissue. Statistical differences in the normalised PrP mRNA levels were found among the tissues, obtaining the highest expression level in obex, followed by ileum, lymph node, spleen, cerebellum and cerebrum. A tendency towards increased PrP mRNA levels and genetic susceptibility was observed in central nervous system. However, the results did not support the hypothesis that PrP mRNA levels vary between genotypes. Conclusion The results on PrP gene expression presented here provide valuable baseline data for future studies on scrapie pathogenesis. On the other hand, the results on stability data of several HK genes reported in this study could prove very useful in other gene expression studies carried out in these relevant ovine tissues.

  8. Validation of Zebrafish (Danio rerio) Reference Genes for Quantitative Real-time RT-PCR Normalization

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    Rongying TANG; Andrew DODD; Daniel LAI; Warren C.MCNABB; Donald R.LOVE

    2007-01-01

    The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The β-actin, EF1α and Rpl13α genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1α, Rpl13α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.

  9. Quantitative real-time RT-PCR and chromogenic in situ hybridization

    DEFF Research Database (Denmark)

    Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia G T;

    2009-01-01

    . 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...

  10. Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei

    Science.gov (United States)

    2005-10-01

    1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions

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

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    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.

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

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    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.

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

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    Huang, S-H; Tsai, M-H; Lin, C-W [Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung, Taiwan (China); Yang, T-C; Chuang, P-H [Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan (China); Tsai, I-S; Lu, H-C [Nanotechnology Research Center, Feng Chia University, Taichung, Taiwan (China); Wan Lei; Lin, Y-J [Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Lai, C-H [Department of Microbiology and Immunology, China Medical University, Taichung, Taiwan (China)], E-mail: cwlin@mail.cmu.edu.tw

    2008-10-08

    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. Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG® RVP fast assay for the detection of respiratory viruses.

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    Choudhary, Manohar L; Anand, Siddharth P; Tikhe, Shamal A; Walimbe, Atul M; Potdar, Varsha A; Chadha, Mandeep S; Mishra, Akhilesh C

    2016-01-01

    Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay.

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

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    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).

  16. Seasonal variation in transcript abundance in cork tissue analyzed by real time RT-PCR.

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    Soler, Marçal; Serra, Olga; Molinas, Marisa; García-Berthou, Emili; Caritat, Antònia; Figueras, Mercè

    2008-05-01

    The molecular processes underlying cork biosynthesis and differentiation are mostly unknown. Recently, a list of candidate genes for cork biosynthesis and regulation was made available opening new possibilities for molecular studies in cork oak (Quercus suber L.). Based on this list, we analyzed the seasonal variation in mRNA abundance in cork tissue of selected genes by real time reverse-transcriptase polymerase chain reaction (RT-PCR). Relative transcript abundance was evaluated by principal component analysis and genes were clustered in several functional subgroups. Structural genes of suberin pathways such as CYP86A1, GPAT and HCBT, and regulatory genes of the NAM and WRKY families showed highest transcript accumulation in June, a crucial month for cork development. Other cork structural genes, such as FAT and F5H, were significantly correlated with temperature and relative humidity. The stress genes HSP17.4 and ANN were strongly positively correlated to temperature, in accord with their protective role.

  17. Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).

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    Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua

    2013-09-15

    Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression.

  18. Quantitative real-time RT-PCR in sentinel lymph nodes from melanoma patients. Detection of melanocytic mRNA predicts disease-free survival

    DEFF Research Database (Denmark)

    Riber-Hansen, Rikke; Abrahamsen, Helene Nortvig; Sorensen, Boe Sandahl

    2008-01-01

    Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) for specific melanoma markers is more sensitive than histology for detecting cells of melanocytic origin in sentinel lymph nodes (SLNs) in cutaneous melanoma. The clinical significance of a positive qRT-PCR analysis...... that the presence of submicroscopic metastases may influence prognosis, indicating that RT-PCR detection of melanocytic cells in SLNs may be an important diagnostic marker....

  19. Detection of Schmallenberg virus in different Culicoides spp. by real-time RT-PCR.

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    De Regge, N; Deblauwe, I; De Deken, R; Vantieghem, P; Madder, M; Geysen, D; Smeets, F; Losson, B; van den Berg, T; Cay, A B

    2012-12-01

    To identify possible vectors of Schmallenberg virus (SBV), we tested pools containing heads of biting midges (Culicoides) that were caught during the summer and early autumn of 2011 at several places in Belgium by real-time RT-PCR. Pools of heads originating from following species: C. obsoletus complex, C. dewulfi and C. chiopterus were found positive, strongly indicating that these species are relevant vectors for SBV.

  20. A quantitative real-time RT-PCR assay for mature C. albicans biofilms

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    Dongari-Bagtzoglou Anna

    2011-05-01

    Full Text Available Abstract Background Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxyanilide (XTT assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells. Results The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay. Conclusions We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures.

  1. Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

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    Piyathida Pongsiri; Kesmanee Praianantathavorn; Apiradee Theamboonlers; Sunchai Payungporn; Yong Poovorawan

    2012-01-01

    ABSTRACT Objective:To develop diagnostic test for detection chikungunya virus (CHIKV and Dengue virus (DENV)infection.Methods:We have performed a rapid, accurate laboratory confirmative method to simultaneously detect, quantify and differentiateCHIKV and DENV infection by single-step multiplex real-timeRT-PCR.Results: The assay’s sensitivity was97.65%, specificity was 92.59% and accuracy was95.82% when compared to conventional RT-PCR. Additionally, there was no cross-reaction betweenCHIKV, DENV, Japanese encephalitis virus, hepatitis C, hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis ofCHIKV andDENV in a single-step reaction.

  2. Comparison of a nucleoprotein gene based RT-PCR with real time RT-PCR for diagnosis of avian influenza in clinical specimens.

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    Nagarajan, S; Murugkar, H V; Tosh, C; Behera, P; Khandia, R; Jain, R; Katare, M; Syed, Z; Tripati, S; Dubey, S C

    2012-08-01

    A nucleoprotein (NP) gene based reverse transcription polymerase chain reaction (npRT-PCR) assay was developed in our laboratory which could detect 35.09% of the experimental samples negative for virus isolation in first passage but positive by third passage. Reducing the reaction volume to 12.5 μl did not alter the test sensitivity and the results did not vary when duplicate samples were run in a different thermal cycler. The positive and negative agreements of this test in clinical specimens were compared with a matrix gene based real time RT-PCR with virus isolation as standard. A total of 516 clinical specimens including tissues, swabs and feces submitted from various States of India as part of active surveillance for avian influenza were tested by npRT-PCR, RRT-PCR and virus isolation in 9-11 day old embryonated specific pathogen free chicken eggs. The positive and negative agreements of npRT-PCR with virus isolation were found to be 0.909±0.022 and 0.980±0.004 respectively and that of RRT-PCR with virus isolation were 0.902±0.023 and 0.977±0.005 respectively. Since the positive and negative agreements of both npRT-PCR and RRT-PCR tests were similar, we suggest that this test can be used by peripheral veterinary laboratories that do not have real time PCR facility for active surveillance of AIV.

  3. Expression patterns of Doppel gene in golden hamster: quantification using real-time RT-PCR.

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    Li, Y R; Li, Q; Yang, J M; Zhou, X M; Yin, X M; Zhao, D M

    2008-08-01

    Doppel (prion-like protein, Dpl) may act as a useful molecular marker in tumor diagnosis and in tumor grade definition, as over-expression of Dpl protein has been found in tumors with different histologic origin. Accordingly, the quantitative analysis of the expression of Dpl in different tissues is essential for understanding its role in tumor progression and cancer diagnostic. Herein we report Dpl mRNA quantification in golden hamster by calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different organs of golden hamster in different stages of development: from neonatal to adult golden hamster. Highest level of Dpl mRNA was detected in the testis, and lower levels of Dpl mRNA were detected in the following tissues: spleen, heart, bone marrow, skeletal muscles and neocortex (only in neonatal hamster). The expression of Dpl was not detected in kidney, liver and lung. This is the first study to report the expression of Dpl in bone marrow of murine and the difference of expression levels of Dpl in testis between adult and neonatal hamsters.

  4. Real time RT-PCR assay for detection of different serotypes of FMDV in Egypt

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    Laila El-Shehawy

    Full Text Available Aim: The present study indicated that rRT-PCR could be provided for the detection of FMDV in infected, contact and carrier cattle and also provide a rapid sensitive tool aiming to aid in rapid disease detection and control. Foot and Mouth disease virus serotypes O and A still existing in Egypt. In January 2012, sever outbreaks struck the animal population in most Egyptian 1 governorates. The causative virus was identified as FMDV SAT2. Material and Methods: Five samples of tongue epithelium (ET and five oesophageal-pharyngeal (OP fluid samples were collected from FMD suspected cattle in infected farm at El-Fayoum and 20 OP samples from in-contact cattle at the same farm in addition to 30 OP samples from apparently healthy cattle at three different localities in El-Fayoum governorate (12 from Fayoum; 9 from Sinoras and 9 from Edsa in order to detect carrier cattle. All of these samples were collected during November and December 2011 and January 2012. Results: All the ET and OP samples were inoculated on BHK cell culture and baby mice. The obtained results were identified using complement fixation test in addition to real-time reverse transcriptase polymerase chain reaction (rRT-PCR. In the infected farm at El-Fayoum FMDV type SAT2 was detected in cattle which are considered as the first introduction of this type while FMDV type O and SAT2 were detected in the in-contact cattle in the same farm. The sensitivity of rRT-PCR was cleared in the in-contact cattle as 13 out of 20 OP samples were positive to FMDV by rRT-PCR while 11 out of 20 OP samples were positive to FMDV by CFT. The OP samples collected from apparently healthy cattle from Fayoum, Sinoras and Edsa localities in Fayoum governorate demonstrate the circulation of the FMDV type A, O and the recent SAT2 in carrier cattle which threaten cattle population in Fayoum governorate. Also the sensitivity of real time RT-PCR over the CFT in detection of FMDV carrier cattle was clearly noticed in

  5. Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

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    Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

    2017-03-01

    Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Quantification of llama inflammatory cytokine mRNAs by real-time RT-PCR.

    Science.gov (United States)

    Odbileg, Raadan; Konnai, Satoru; Usui, Tatsufumi; Ohashi, Kazuhiko; Onuma, Misao

    2005-02-01

    We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF) alpha were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1alpha, IL-1beta, IL-6 and TNFalpha were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.

  7. Quantitation of Rabbit Cytokine mRNA by Real-Time RT-PCR

    OpenAIRE

    Godornes, Charmie; Leader, Brandon Troy; Molini, Barbara J.; Centurion-Lara, Arturo; Lukehart, Sheila A.

    2007-01-01

    Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-γ, IL-2, IL-4, IL-10 and TNF-α mRNA. Quantitation was accomplished by comparison to a st...

  8. Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis.

    Science.gov (United States)

    Rocha-Silva, Fabiana; Gomes, Luciana I; Gracielle-Melo, Cidiane; Goes, Alfredo M; Caligiorne, Rachel B

    2017-01-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological

  9. Real time TaqMan RT-PCR assay for the detection of Cucumber green mottle mosaic virus.

    Science.gov (United States)

    Hongyun, Chen; Wenjun, Zhao; Qinsheng, Gu; Qing, Chen; Shiming, Lin; Shuifang, Zhu

    2008-05-01

    A real time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of Cucumber green mottle mosaic virus (CGMMV). The method was designed to use a duo-primer system with a TaqMan probe targeting the conserved sequence in 3' noncoding region (NCR) of CGMMV to detect isolates of this virus collected in China. The sensitivity of the real time RT-PCR assay was 0.13 pg of total RNA or 50 molecules of RNA transcripts. This level of sensitivity indicated that the one step real time RT-PCR developed in the present study could be used for routine testing assays. The real time RT-PCR method could assist in the implementation of quarantine measures for prevention and control of the disease caused by CGMMV.

  10. Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes

    Directory of Open Access Journals (Sweden)

    Yi-Hong Zhou

    2010-08-01

    Full Text Available In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients—a process that requires large sample sizes by combining independent sets of data.

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

    Science.gov (United States)

    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.

  12. Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

    Directory of Open Access Journals (Sweden)

    Barredo Julio C

    2002-03-01

    Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

  13. A duplex real-time RT-PCR assay for profiling inhibitors of four dengue serotypes.

    Science.gov (United States)

    Gong, Edwin Yunhao; Smets, Alexandra; Verheyen, Nick; Clynhens, Marleen; Gustin, Emmanuel; Lory, Pedro; Kraus, Guenter

    2013-01-01

    We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four dengue serotypes deposited in the GeneBank. To discriminate the antiviral activity from the cytotoxicity of compounds, a housekeeping gene of the Vero cells, β-actin, was used to design the primers and the probe for the second set of PCR as an internal control, which is used to normalize the RNA levels of dengue-specific PCR due to the cellular toxicity of test compounds. For compound profiling, the duplex PCR is performed using LightCycler(®) in a single tube to simultaneously amplify both the dengue target gene and the Vero cell housekeeping gene from the compound-treated Vero cell lysates. This assay was validated against a panel of reference compounds. The results show that the universal primers and probe in this duplex RT-PCR assay can efficiently amplify all four dengue serotypes and that the PCR efficiency for both the dengue target gene and the Vero cells β-actin gene is 100%.

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

    Science.gov (United States)

    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.

  15. Cytokine mRNA quantification in histologically normal canine duodenal mucosa by real-time RT-PCR.

    Science.gov (United States)

    Peters, I R; Helps, C R; Calvert, E L; Hall, E J; Day, M J

    2005-01-10

    CD4(+) T helper cells are important for the regulation of immune responses in the intestinal mucosa and they exert their effects through the secretion of pro-inflammatory and immunomodulatory cytokines. Human patients with inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have alterations in the normal intestinal cytokine profile. These cytokine abnormalities have been shown at both the protein and messenger RNA (mRNA) level. The role that mucosal cytokines play in the pathogenesis of canine IBD has only been investigated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of gut tissue, as cytokine antisera are not available for this species. Real-time RT-PCR has been recognised to be a more accurate and sensitive method of quantifying mRNA transcripts, so in this study TaqMan real-time RT-PCR assays for the quantification of mRNA encoding IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IFN-gamma, TNF-alpha and TGF-beta in canine intestinal mucosa were developed. The amount of these templates was quantified in normal canine duodenal mucosa (n = 8). IL-18, TGF-beta and TNF-alpha were found to be the most abundant transcripts, with IL-10 and IFN-gamma present at levels approximately 10-fold less. IL-2, IL-4, IL-5, IL-6 and IL-12 were the least abundant templates, with some RNA samples having no detectable mRNA copies. The methods developed in this study will form the basis of further work investigating the expression of mRNA encoding cytokines in mucosa from dogs with chronic enteropathies. In addition, these real-time PCR assays can also be used for the quantification of canine cytokine mRNA in other diseases.

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

    Directory of Open Access Journals (Sweden)

    Borges-Pérez Andrés

    2008-12-01

    Full Text Available Abstract 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

  17. Improved Serotype-Specific Dengue Virus Detection in Trinidad and Tobago using a Multiplex, Real-Time RT-PCR

    Science.gov (United States)

    Waggoner, Jesse J.; Sahadeo, Nikita S. D.; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V. F.; Pinsky, Benjamin A.

    2014-01-01

    Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance is limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time RT-PCR detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; p=0.01). PMID:25533614

  18. The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

    NARCIS (Netherlands)

    Clancy, A.; Crowley, B.; Niesters, H.; Herra, C.

    2008-01-01

    Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM 7500 Sequence Detection

  19. The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

    NARCIS (Netherlands)

    Clancy, A.; Crowley, B.; Niesters, H.; Herra, C.

    2008-01-01

    Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM 7500 Sequence Detection

  20. Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

    Science.gov (United States)

    Rupprom, Kitwadee; Chavalitshewinkoon-Petmitr, Porntip; Diraphat, Pornphan; Kittigul, Leera

    2017-02-20

    Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10(3) DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

  1. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.

    Science.gov (United States)

    Nicot, Nathalie; Hausman, Jean-François; Hoffmann, Lucien; Evers, Danièle

    2005-11-01

    Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes (beta-tubulin, cyclophilin, actin, elongation factor 1-alpha (ef1alpha), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1alpha was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

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

    Science.gov (United States)

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

    2013-07-01

    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.

  3. Real-time RT-PCR expression analysis of chitinase and endoglucanase genes in the three-way interaction between the biocontrol strain Clonostachys rosea IK726, Botrytis cinera and strawberry

    DEFF Research Database (Denmark)

    Mamarabadi, Mojtaba; Jensen, Birgit; Jensen, Søren Dan Funck

    2008-01-01

    Clonostachys rosea is a well-known biocontrol agent against Botrytis cinerea, the causal agent of gray mold in strawberry. The activity of cell wall-degrading enzymes might play a significant role for successful biocontrol by C. rosea. The expression pattern of four chitinases, and two...... endoglucanase genes from C. rosea strain IK726 was analyzed using real-time RT-PCR in vitro and in strawberry leaves during interaction with B. cinerea. Specific primers were designed for ß-tubulin genes from C. rosea and B. cinerea, respectively, and a gene encoding a DNA-binding protein (DBP) from strawberry......, allowing in situ activity assessment of each fungus in vitro and during their interaction on strawberry leaves. Growth of B. cinerea was inhibited in all pathogen-antagonist interactions while the activity of IK726 was slightly increased. In all in vitro interactions, four of the six genes were upregulated...

  4. Assessment of HER-2 gene overexpression in Isfahan province breast cancer patients using Real Time RT-PCR and immunohistochemistry.

    Science.gov (United States)

    Tabatabaeian, Hosein; Hojati, Zohreh

    2013-11-15

    Overexpression of proto-oncogene HER-2 is one of the main molecular markers of breast cancer involved in prognosis and diagnosis and also in trastuzumab therapy. Thus, a request for the evaluation of HER-2 status in breast cancer has been increasing. The aim of our study was assessment of HER-2 overexpression in malignant and benign breast cancer specimens by Real Time RT-PCR technique and comparison of its results with IHC outcomes. Twenty benign and sixty malignant breast cancers in addition to fifteen normal breast tissue specimens were analyzed by Real Time RT-PCR method. Fresh tissue samples were disrupted by mortar and pestle. A syringe and a needle were used for complete homogenization of the tissues. The RNA was then isolated from the samples and converted to cDNA. A standard curve was initially plotted using BioEasy SYBR Green I and then all 95 specimens were studied by Real Time RT-PCR using 2(-ΔΔCt) method. 23.3% of 60 malignant specimens showed HER-2 overexpression, while all of the benign samples represented the normal expression level of HER-2 gene. The concordance rate between the results of Real Time RT-PCR and IHC was 86.6%. Real Time RT-PCR method is an almost reliable technique and at least can be used as a complementary method for confirming IHC results. This is emanated from relatively high rate of concordance between outcomes of IHC test, as a routine method of detecting the HER-2 gene expression status, and Real Time RT-PCR technique. © 2013 Elsevier B.V. All rights reserved.

  5. Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR.

    Science.gov (United States)

    Abrahamian, Peter E; Seblani, Rewa; Sobh, Hana; Abou-Jawdah, Yusuf

    2013-11-01

    Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) are whitefly-transmitted criniviruses infecting cucurbit crops inducing similar symptoms. Single and multiplex RT-PCR protocols were developed and evaluated on cucurbit samples collected from commercial greenhouses. Primers and probes were designed from the highly conserved heat shock protein 70 homolog (Hsp70h) gene. Conventional RT-PCR and multiplex RT-PCR assays showed high specificity and suitability for routine screening. TaqMan-based quantitative real-time RT-PCR (RT-qPCR) protocols were also developed for the detection and quantitation of both viruses occurring in single or mixed infection. The assays proved to be highly specific with no cross amplification. RT-qPCR assays showed a 100-1000 times improved sensitivity over conventional RT-PCR. Virus titers in mixed infections were compared to singly infected plants by RT-qPCR. CYSDV and CCYV titers decreased in double infected plants. This paper reports highly specific conventional RT-PCR and quantitative real-time PCR assays for detection, quantitation and differentiation between two closely related cucurbit-infecting criniviruses.

  6. The development and application of the two real-time RT-PCR assays to detect the pathogen of HFMD.

    Directory of Open Access Journals (Sweden)

    Aili Cui

    Full Text Available Large-scale Hand, Foot, and Mouth Disease (HFMD outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs. Among them, human enterovirus 71 (HEV71 and coxsackievirus A16 (CVA16 are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1-2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID50/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells. The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11 by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.

  7. Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.

    Directory of Open Access Journals (Sweden)

    Stephanie L Angione

    Full Text Available As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR. Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic, H1N1s (seasonal, and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 10(4 copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies.

  8. Meloidogyne javanica Chorismate Mutase Transcript Expression Profile Using Real-Time Quantitative RT-PCR.

    Science.gov (United States)

    Painter, Janet E; Lambert, Kris N

    2003-03-01

    A developmental expression profile of the Meloidodgyne javanica esophageal gland gene chorismate mutase-1 (Mj-cm-1) could suggest when in the lifecycle of the nematode the Mj-cm-1 product is functional. This study used real-time quantitative RT-PCR to examine the variation in Mj-cm-1 transcript levels over six timepoints in the nematode lifecycle: egg, infective second-stage juveniles (Inf-J2), 2-day post-inoculation (pi), 7-day pi, 14-day pi, and adult. The Mj-cm-1 mRNA levels peaked at 2-day pi, about 100-fold above levels expressed at the egg and Inf-J2 stages. Some expression of Mj-cm-1 remained during the 7-day pi, 14-day pi, and adult stages. High transcript levels of the beta-actin control gene M. javanica Beta-actin-1 (Mj-ba-1) demonstrated the presence of cDNA at all timepoints. The peak in Mj-cm-1 transcript expression at 2-day pi as well as the previously shown esophageal gland localization of Mj-cm-1 mRNA suggest that the product of this gene may be involved early in the establishment of parasitism.

  9. RT-PCR and real-time RT-PCR methods for the detection of potato virus Y in potato leaves and tubers.

    Science.gov (United States)

    MacKenzie, Tyler D B; Nie, Xianzhou; Singh, Mathuresh

    2015-01-01

    Potato virus Y (PVY) is a major threat to potato crops around the world. It is an RNA virus of the family Potyviridae, exhibiting many different strains that cause a range of symptoms in potato. ELISA detection of viral proteins has traditionally been used to quantify virus incidence in a crop or seed lot. ELISA, however, cannot reliably detect the virus directly in dormant tubers, requiring several weeks of sprouting tubers to produce detectable levels of virus. Nor can ELISA fully discriminate between the wide range of strains of the virus. Several techniques for directly detecting the viral RNA have been developed which allow rapid detection of PVY in leaf or tuber tissue, and that can be used to easily distinguish between different strains of the virus. Described in this chapter are several protocols for the extraction of RNA from leaf and tuber tissues, and three detection methods based upon reverse-transcription-PCR (RT-PCR). First described is a traditional two-step protocol with separate reverse transcription of viral RNA into cDNA, then PCR to amplify the viral cDNA fragment. Second described is a one-step RT-PCR protocol combining the cDNA production and PCR in one tube and one step, which greatly reduces material and labor costs for PVY detection. The third protocol is a real-time RT-PCR procedure which not only saves on labor but also allows for more precise quantification of PVY titre. The three protocols are described in detail, and accompanied with a discussion of their relative advantages, costs, and possibilities for cost-saving modifications. While these techniques have primarily been developed for large-scale screening of many samples for determining viral incidence in commercial fields or seed lots, they are also amenable to use in smaller-scale research applications.

  10. Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus.

    Science.gov (United States)

    Zhou, Xinrong; Zhang, Tiansheng; Song, Deping; Huang, Tao; Peng, Qi; Chen, Yanjun; Li, Anqi; Zhang, Fanfan; Wu, Qiong; Ye, Yu; Tang, Yuxin

    2017-06-01

    Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal disease, resulting in substantial economic losses to the swine industry worldwide. In this study, three assays, namely a conventional reverse transcription-polymerase chain reaction (RT-PCR), a SYBR Green I real-time RT-PCR and a TaqMan real-time RT-PCR targeting the highly conserved M gene of PEDV, were developed and evaluated. Then, the analytical specificity, sensitivity and reproducibility of these assays were determined and compared. The TaqMan real-time RT-PCR was 100-fold and 10,000-fold more sensitive than that of the SYBR Green I real-time RT-PCR and the conventional RT-PCR, respectively. The analytical sensitivity of TaqMan real-time RT-PCR was 10 copies/μl of target gene and no cross amplification with other viruses tested was observed. With the features of high specificity, sensitivity, and reproducibility, the TaqMan real-time RT-PCR established in this study could be a useful tool for clinical diagnosis, epidemiological surveys and outbreak investigations of PED. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR

    Directory of Open Access Journals (Sweden)

    Lou Sai

    2011-07-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV is a worldwide pathogen in cattle and acts as a surrogate model for hepatitis C virus (HCV. One-step real-time fluorogenic quantitative reverse transcription polymerase chain reaction (RT-PCR assay based on SYBR Green I dye has not been established for BVDV detection. This study aims to develop a quantitative one-step RT-PCR assay to detect BVDV type-1 in cell culture. Results One-step quantitative SYBR Green I RT-PCR was developed by amplifying cDNA template from viral RNA and using in vitro transcribed BVDV RNA to establish a standard curve. The assay had a detection limit as low as 100 copies/ml of BVDV RNA, a reaction efficiency of 103.2%, a correlation coefficient (R2 of 0.995, and a maximum intra-assay CV of 2.63%. It was 10-fold more sensitive than conventional RT-PCR and can quantitatively detect BVDV RNA levels from 10-fold serial dilutions of titrated viruses containing a titer from 10-1 to 10-5 TCID50, without non-specific amplification. Melting curve analysis showed no primer-dimers and non-specific products. Conclusions The one-step SYBR Green I RT-PCR is specific, sensitive and reproducible for the quantification of BVDV in cell culture. This one-step SYBR Green I RT-PCR strategy may be further optimized as a reliable assay for diagnosing and monitoring BVDV infection in animals. It may also be applied to evaluate candidate agents against HCV using BVDV cell culture model.

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

    Science.gov (United States)

    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...

  13. Measurement of bacterial gene expression in vivo by laser capture microdissection and quantitative real-time RT-PCR

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Jensen, Tim Kåre; Angen, Øystein

    2007-01-01

    Due to the relative small number of bacterial pathogens present in an infected host, exploration of pathogen gene expression in vivo is challenging. This study reports the development of a protocol for quantifying bacterial gene expression in vivo in Actinobacillus pleuropneumoniae using laser ca...... capture microdissection and real-time quantitative RT-PCR....

  14. Real-time Quantitative RT-PCR for CT9 Level in Human Cancer

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    CT9 is a recently cloned cancer-testis antigen, which is a member of the bromodomain and extraterminal family.Each member of this protein family contains two N-terminal bromodomain motifs. We investigated the distribution of CT9 in different tissues and the possibility for it to be used as a potential therapeutic target in cancer treament. By using the real-time RT-PCR method and 18SrRNA as an internal standard, we analyzed the CT9 expression in several normal human tissues and in the tissues of patients suffering from cancer. The result of this study shows that the highest level of mRNA is only present in testis tissue because the CT9 expression has not been detected in other normal tissues. In 6 of 10 cases of gastric adenocarcinoma, in 3 of 10 cases of esophageal squamous cell carcinoma, in 2 of 9 cases of endometrial carcinoma and only in 1 of 12 cases of brain cancer, the low level expression of CT9 was detected. In none of the 12 cases of cervical squamous cell carcinoma, the expression of CT9 was detected. Since the high level expression of CT9 is only found in the normal testis tissue, but the low expression in cancer tissues, for example tissues of cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma, we conclude that CT9 cannot be used as a cancer therapeutic target molecule for cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma.

  15. Identification of nasal blood by real-time RT-PCR.

    Science.gov (United States)

    Sakurada, Koichi; Akutsu, Tomoko; Watanabe, Ken; Yoshino, Mineo

    2012-07-01

    A new approach for the identification of body fluid stains by comparing specific mRNA expression levels has been extensively studied in recent years. Here, we examine whether nasal blood, which is regarded as one of the most difficult types of blood to identify, can be identified by comparing mRNA expression levels of target genes specific to saliva, nasal secretion, and blood. The saliva-specific statherin gene (STATH) was found to be expressed at high levels in not only saliva (dCt value: 1.32±1.39, n=5), but also nasal secretions (dCt value: 0.90±1.14, n=5), while the histatin gene (HTN3) was only expressed at high levels in saliva (dCt value: 1.08±2.35, n=5). We also confirmed that the hemoglobin-beta gene (HBB) showed high expression levels in blood (dCt value: -9.51±0.40, n=5). Four nasal blood stains were found to highly express STATH (dCt value: 5.65±3.98) and HBB (dCt value: -8.79±1.67) but not HTN3, suggesting that the stain samples contained both nasal secretions and blood and can therefore be identified as nasal blood stains. Although menstrual blood showed the same expression pattern as nasal blood, the menstrual blood-specific protein matrix metallopeptidase 7 (MMP7) was not expressed in all nasal blood stain samples. Therefore, its expression levels could be used to discriminate between nasal and menstrual blood. In conclusion, real-time RT-PCR was able to identify nasal blood, although the stability of gene expression in nasal blood stains was low over time, suggesting that this assay may not be effective for older stains. Future work should examine the usefulness of this assay under various environmental conditions.

  16. Role of real-time PCR (RT-PCR) in rapid diagnosis of tuberculous mycobacteria in different clinical samples.

    Science.gov (United States)

    2014-02-01

    The study was aimed for molecular detection of mycobacterial DNA in different clinical samples using real-time polymerase chain reaction (RT-PCR) system and rapid diagnosis of tuberculosis. A total of 508 clinical specimens (blood 343, menstrual fluid 53, endometrial tissue 43, body fluid 36, pus from lymph nodes 18, sputum 8, urine 5 and semen 2) were included in this study. We extracted DNA using QIAamp DNA Mini Kit (QIAGEN, Germany) and performed real-time assay using Rotor-Gene Q machine from Corbett Research, Australia for specific amplification of IS6110 sequence of mycobacterial genome. The RT-PCR result was also compared with bacterial culture and acid-fast bacillus staining. RT-PCR assay showed positivity in 52 cases and negative in 456 cases. Corresponding positive results in culture and acid-fast bacillus staining methods were 49 cases and 24 cases respectively. The sensitivity and specificity of detecting Mycobacterium tuberculosis by RT-PCR were 93.87% and 98.69% respectively taking positive culture results as reference standards. The overall positive and negative predictive values were 88.46% and 99.34% respectively. RT-PCR is a useful diagnostic tool for rapid and sensitive detection of mycobacteria in different clinical samples. The easy processing, fast reporting and relative lack of contamination issues make it worthy as a possible replacement to time consuming culture techniques. Moreover, it has added advantage of quantification of mycobacterial DNA, hence bacterial load.

  17. Development of real-time RT-PCR for the detection of low concentrations of Rift Valley fever virus.

    Science.gov (United States)

    Maquart, Marianne; Temmam, Sarah; Héraud, Jean-Michel; Leparc-Goffart, Isabelle; Cêtre-Sossah, Catherine; Dellagi, Koussay; Cardinale, Eric; Pascalis, Hervé

    2014-01-01

    In recent years, Madagascar and the Comoros archipelago have been affected by epidemics of Rift Valley fever (RVF), however detection of Rift Valley fever virus (RVFV) in zebu, sheep and goats during the post epidemic periods was frequently unsuccessful. Thus, a highly sensitive real-time RT-PCR assay was developed for the detection of RVFV at low viral loads. A new RVF SYBR Green RT-PCR targeting the M segment was tested on serum from different RVF seronegative ruminant species collected from May 2010 to August 2011 in Madagascar and the Comoros archipelago and compared with a RVF specific quantitative real time RT-PCR technique, which is considered as the reference technique. The specificity was tested on a wide range of arboviruses or other viruses giving RVF similar clinical signs. A total of 38 out of 2756 serum samples tested positive with the new RT-PCR, whereas the reference technique only detected 5 out of the 2756. The described RT-PCR is an efficient diagnostic tool for the investigation of enzootic circulation of the RVF virus. It allows the detection of low viral RNA loads adapted for the investigations of reservoirs or specific epidemiological situations such as inter-epizootic periods.

  18. Application of real-time RT-PCR quantification to evaluate differential expres sion of Arabidopsis Aux/IAAgenes

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The molecular techniques including Northern blot, dot blot, in situ hybridization, etc. have been success fully used to estimate semi-quantitatively mRNA levels in plant samples. In this study, we employed a real-time reverse transcription-PCR (RT-PCR) assay using SYBR Green Ifluorescence methodology to evaluate accurate quant itation and sequence specific detection of Aux/IAA mRNA levels in Arabidopsis. Results obtained indicate a linear dynamic range of 102-106 Aux/IAA mRNA copies with standard de viations of generally less than 15%. As a model experiment,the outcome of analysis of expression patterns of five Aux/IAA genes in Arabidopsis under various chemical and temperature treatments is presented. The method presented here provides a sensitive and rapid technique to evaluate plant Aux/IAA mRNA expression levels in nanogram order.

  19. Processing of gene expression data generated by quantitative real-time RT-PCR.

    Science.gov (United States)

    Muller, Patrick Y; Janovjak, Harald; Miserez, André R; Dobbie, Zuzana

    2002-06-01

    Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Comparison of real time RT-PCR and flow cytometry methods for evaluation of biological activity of recombinant human erythropoietin

    Directory of Open Access Journals (Sweden)

    Sepehrizadeh Z

    2008-05-01

    Full Text Available Background: Evaluation of bioactivity of recombinant erythropoietin is essential for pharmaceutical industry, quality control authorities and researchers. The purpose of this study was to compare real time RT-PCR and flow cytometry for the assay of biological activity of recombinant erythropoietin. Methods: Three concentrations of recombinant erythropoietin BRP (80, 40 and 20 IU/ml were injected subcutaneously to mice. After 4 days the blood was collected and used for reticulocyte counts by flow cytometry and also for the RNA extraction. Real time RT-PCR amplification was carried out for β-globin. Results and conclusion: There was a significant correlation between the total RNA amounts (R2= 0.9995, relative quantity of β-globin mRNA (R2= 0.984 and reticulocyte counts (R2= 0.9742 with rhEpo concentrations. Total RNA and quantitative RT-PCR showed significant dose dependent results as well the reticulocyte counts by flow cytometry for the biological activity assay of rhEpo and so these methods could be considered as alternatives for flow cytometry.

  3. Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses.

    Science.gov (United States)

    Cecilia, D; Kakade, M; Alagarasu, K; Patil, J; Salunke, A; Parashar, D; Shah, P S

    2015-01-01

    Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.

  4. Real time polymerase chain reaction (rt-pcr for mycobacterium tuberculosis in serpiginous choroiditis- A study of 29 cases

    Directory of Open Access Journals (Sweden)

    RadhaAnnamalai, Jyotirmay Biswas, Sudharshan S, Gayathri R, Lily Therese, Viswanathan S, NamithaBhuvaneswari

    2014-07-01

    Full Text Available Purpose: A study of real time Polymerase Chain Reaction for Mycobacterium tuberculosis (M. tuberculosis DNA in 29 cases of active serpiginous choroiditis. Design: Case control study. Methods: DNA extraction from the aqueous humor was carried out using QIAMP DNA extraction kit. Real- time Polymerase Chain reaction (RT-PCR for MTB was carried out using Genosen’s Mtb complex quantitative Real time PCR kit. All patients were also subjected to complete blood count, venereal disease research laboratory test, chest radiograph, QuantiFERON TB Gold test on the blood and polymerase chain reaction on a sample of aqueous humor. Results: Aqueous aspirate showed copies of mycobacterium tuberculosis DNA in one out of twenty nine cases of serpiginous choroiditis. Direct smear and culture for mycobacteria was negative in all cases. Conclusion: RT-PCR identifies MTB DNA in suspected latent tuberculosis in serpiginous choroiditis with high specificity. Serpiginous choroiditis and multifocal choroiditis due to tuberculosis may resemble each other clinically but have distinct clinical features which can be confirmed by real time polymerase chain reaction performed on the aqueous humor The association between serpiginous choroiditis and tuberculosis would be a chance association or if present a rare association.

  5. Development and implementation of the quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1 surveillance network in mainland China

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2011-03-01

    Full Text Available Abstract Background Reverse transcription PCR (RT-PCR and real time RT-PCR (rRT-PCR have been indispensable methods for influenza surveillance, especially for determination of avian influenza. The movement of testing beyond reference lab introduced the need of quality control, including the implementation of an evaluation system for validating personal training and sample proficiency testing. Methods We developed a panel with lysates of seasonal influenza virus (H1N1, H3N2 and B, serials of diluted H5N1 virus lysates, and in-vitro transcribed H5 hemaglutinin (HA and an artificial gene RNAs for RT-PCR and rRT-PCR quality control assessment. The validations of stability and reproducibility were performed on the panel. Additionally, the panel was implemented to assess the detection capability of Chinese human avian influenza networks. Results The panel has relatively high stability and good reproducibility demonstrated by kappa's tests. In the implementation of panel on Chinese human avian influenza networks, the results suggested that there were a relatively low number of discrepancies for both concise and reproducibility in Chinese avian influenza virus net works. Conclusions A quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1 surveillance network was developed. An availably statistical data, which are used to assess the detection capability of networks on avian influenza virus (H5N1, can be obtained relatively easily through implementation of the panel on networks.

  6. Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

    DEFF Research Database (Denmark)

    Lyng, Maria B; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

    2008-01-01

    of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER......- IBC, normal breast tissue and breast cancer cell lines. METHODS: The reference genes investigated by qRT-PCR were RPLP0, TBP, PUM1, ACTB, GUS-B, ABL1, GAPDH and B2M. Biopsies of 18 surgically-excised tissue specimens (11 ER+ IBCs, 4 ER- IBCs, 3 normal breast tissues) and 3 ER+ cell lines were examined...... of human tissue samples (ER+ and ER- IBC and normal breast tissue) and for the invasive cancer samples (ER+ and ER- IBC) by GeNorm, where NormFinder consistently identified PUM1 at the single best gene for all sample combinations. CONCLUSION: The reference genes of choice when performing RT-qPCR on normal...

  7. Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs

    DEFF Research Database (Denmark)

    Reid, S.M.; Paton, D.J.; Wilsden, G.;

    2004-01-01

    of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs....

  8. Quantification of GPCR mRNA using real-time RT-PCR.

    Science.gov (United States)

    Brattelid, Trond; Levy, Finn Olav

    2011-01-01

    Characterisation of G-protein-coupled receptor (GPCR) mRNA expression under normal, different pharmacological and pathological conditions in experimental animal models and human tissue biopsies by quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a valuable approach to understand the regulation of GPCR expression. RT-qPCR is specific and sensitive with a broad dynamic range, which allows precise quantification of mRNA species of interest. In addition to measuring the relative levels of mRNA in a tissue or changes in expression levels between groups of genes of interest, RT-qPCR is also used to identify splice variants and single nucleotide polymorphisms (SNPs) of GPCRs. Even though RT-qPCR has become the standard method for quantification of gene expression, RT-qPCR is sensitive to RNA quality, assay design, normalisation approach and data analysis. This protocol is meant as a guide to RT-qPCR methodology with references to the best standard methods available at present.

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

    Directory of Open Access Journals (Sweden)

    Jin-Guang Yang

    2012-12-01

    Full Text Available 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.

  10. Expression patterns of prion protein gene in differential genotypes sheep: quantification using molecular beacon real-time RT-PCR.

    Science.gov (United States)

    Wang, Chuan; Wu, Run; Li, Fa-Di; Liu, Lei; Zhang, Xiao-Li; Zhao, Chun-Lin; Diao, Xiao-Long; Guan, Hong-Wei

    2011-06-01

    Determination of the transcription level of cellular prion protein (PrP(C)) is essential for understanding its role in organisms and revealing mechanism of susceptibility and resistance to scrapie. However, the expression of prion protein (PrP) mRNA in sheep has not been quantified in great detail in digestive tract which is important during scrapie spread through oral route. Herein, we report on measurement of sheep PrP mRNA using absolute quantitative real-time RT-PCR. Total RNA was isolated from five different regions of the central nervous system (CNS), four regions of lymphoid system, eleven regions of digestive tract, and two reproductive organ tissues of eight sheep of two different genotypes (ARR/ARQ and ARH/ARQ) and PrP mRNA was quantified by real-time RT-PCR using molecular beacon. The results showed that highest levels of PrP mRNA were expressed in thalamus and cerebrum (P mRNA expression in sheep for further studies of pathogenesis of prion diseases.

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

    Directory of Open Access Journals (Sweden)

    Toth Ian K

    2007-09-01

    Full Text Available Abstract 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.

  12. Comprehensive selection of reference genes for gene expression normalization in sugarcane by real time quantitative rt-PCR.

    Directory of Open Access Journals (Sweden)

    Hui Ling

    Full Text Available 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.

  13. Molecular staging of pathologically negative sentinel lymph nodes from melanoma patients using multimarker, quantitative real-time rt-PCR.

    Science.gov (United States)

    Hilari, Josep M; Mangas, Cristina; Xi, Liqiang; Paradelo, Cristina; Ferrándiz, Carlos; Hughes, Steven J; Yueh, Cindy; Altomare, Ivy; Gooding, William E; Godfrey, Tony E

    2009-01-01

    The aim of this study was to evaluate the prognostic potential of quantitative reverse-transcription, polymerase chain reaction (qRT-PCR) in melanoma patients with pathologically negative sentinel lymph nodes (SLN). Our study included 195 node-negative melanoma patients with a Breslow thickness greater than 0.76 mm (n = 158), or less than 0.76 mm but who had Clark level IV-V, microscopic ulceration, or pathological signs of regression (n = 32), and five patients with melanoma of unknown thickness. SLNs were examined by serial-section histopathology. A portion of each SLN was frozen for qRT-PCR analysis using markers Tyrosinase, MART1, SSX2, MAGEA3, PAX3, and GalNAc-T. In addition, two other markers (PLAB and L1CAM) were evaluated for melanoma specificity but not for SLN analysis. Median follow-up was 64 months, during which time there were 15 (7.7%) recurrences. A total of 370 lymph nodes were analyzed by qRT-PCR. No association was found between quantitative expression level of any marker and disease recurrence. Previously published primer designs were tested for PAX3 and GalNAc-T and revealed that alternative PAX3 transcripts are differentially expressed in melanoma and benign lymph nodes. No associations with recurrence were found regardless of the transcripts amplified by different primer sets. PLAB and L1CAM did not appear to differentiate between malignant melanoma and benign melanocytes or lymph nodes in our analysis. We conclude that, in this large cohort of patients, multimarker qRT-PCR analysis of SLNs did not correlate with disease recurrence. Our data support specific PAX3 splice variants but not GalNAc-T, PLAB or L1CAM as possible markers for melanoma metastasis to SLNs.

  14. 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.

    Science.gov (United States)

    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.

  15. Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome.

    Science.gov (United States)

    Polci, A; Cosseddu, G M; Ancora, M; Pinoni, C; El Harrak, M; Sebhatu, T T; Ghebremeskel, E; Sghaier, S; Lelli, R; Monaco, F

    2015-06-01

    A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/μl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection. © 2013 Blackwell Verlag GmbH.

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

    Science.gov (United States)

    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.

  17. Expression patterns of Doppel in differential ovine PRNP genotypes: quantification using real-time RT-PCR.

    Science.gov (United States)

    Wang, C; Zhao, C-L; Liu, L; Wu, R; Zhang, X-L

    2015-10-09

    Doppel is a homologue of cellular prion protein (PrP)-like protein (PrPC). Different tissue samples were collected from the central nervous system plus four regions of lymphoid system, eleven regions of digestive tract and two reproductive organs from four ARR/ARQ and four ARH/ARQ sheep, genotypes of the PrP gene. Total RNA was isolated from these samples, and Doppel mRNA was quantified by real-time RT-PCR using SYBR Green. Doppel mRNA expression was higher in the ovary, hypothalamus and brain than in other tissues, and it significantly differed between the two genotypes in brain, ileum, cecum, rectum, colon, and uterus. This study demonstrated that Doppel mRNA expression in sheep with ARR/ARQ or ARH/ARQ genotypes was very different. These findings could be helpful in future studies of the relationship between PrP and Doppel.

  18. Quantification of dissimilatory (bi)sulphite reductase gene expression in Desulfobacterium autotrophicum using real-time RT-PCR

    DEFF Research Database (Denmark)

    Neretin, LN; Schippers, A.; Pernthaler, A.

    2003-01-01

    We developed a real-time RT-PCR method for the quantification of dissimilatory (bi)sulphite reductase (DSR) mRNA in Desulfobacterium autotrophicum cells. The amount of DSR mRNA was determined relative to the amount of 16S rRNA at different growth conditions during transition from exponential...... changed significantly during growth (up to 310-fold from the early to the late exponential phase during respiration with thiosulphate). The maximum DSR mRNA per-cell contents correlated with cell-specific sulphate reduction rates for all experiments. Environmental applications for the quantification...... to stationary phase: sulphate respiration with lactate, thiosulphate respiration with lactate, sulphate respiration with H-2 and pyruvate fermentation. The dsr gene was expressed constitutively, although DSR mRNA content per-cell varied under different growth conditions. The maximum DSR mRNA per-cell content...

  19. Detecting PML-RARα transcript in acute promyelocytic leukemia using real-time quantitative RT-PCR

    Institute of Scientific and Technical Information of China (English)

    ZHU Hong-hu; LIU Yan-rong; QIN Ya-zhen; JIANG Bin; SHAN Fu-xiang; WU Shu-lan; YANG Ping-di; ZHAO Jie; LU Dao-pei

    2007-01-01

    Background Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia. However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t (15; 17) from October 2004 to December 2005.Methods All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7500platform. The quantitation of PML-RARα transcripts was represented by the normalized quotient, that is, PML-RARα transcript copies divided by ABL transcript copies. According to induction therapy, the patients were classed into two groups: group 1 (n=23), three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 (n=13), two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone.Results The sensitivity of RQ-PCR was 1 per 105 cells and 5 copies of the PML-RARα transcript could be reproducibly detected. No false positive results occurred in 40 non-acute promyelocytic leukemia samples. Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves (slope: -3.2 - -3.7). The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 (61 days vs 75 days, P=0.034). The rate of molecular remission within 70days was higher in group 1 than in group 2 (75.00% vs 38.46%, P=0.036). Compared with pretreatment, median reduction of the PML-RARα transcript before first consolidation therapy differed significantly between group 1 and group 2 (log scale, 3.15 vs 2

  20. Selection of normalization factors for quantitative real time RT-PCR studies in Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus) under conditions of viral infection.

    Science.gov (United States)

    Zhang, Jian; Hu, Yong-hua; Sun, Bo-guang; Xiao, Zhi-zhong; Sun, Li

    2013-04-15

    Disease outbreaks caused by iridoviruses are known to affect farmed flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). To facilitate quantitative real time RT-PCR (qRT-PCR) analysis of gene expression in flounder and turbot during viral infection, we in this study examined the potentials of 9 housekeeping genes of flounder and turbot as internal references for qRT-PCR under conditions of experimental infection with megalocytivirus, a member of the Iridoviridae family. The mRNA levels of the 9 housekeeping genes in the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of flounder and turbot were determined by qRT-PCR at 24h and 72h post-viral infection, and the expression stabilities of the genes were determined with geNorm and NormFinder algorithms. The results showed that (i) viral infection induced significant changes in the mRNA levels of the all the examined genes in a manner that was dependent on both tissue type and infection stage; (ii) for a given time point of infection, stability predictions made by the two algorisms were highly consistent for most tissues; (iii) the optimum reference genes differed at different infection time points at least in some tissues; (iv) at both examined time points, no common reference genes were identified across all tissue types. These results indicate that when studying gene expression in flounder and turbot in relation to viral infection, different internal references may have to be used not only for different tissues but also for different infection stages.

  1. Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs

    DEFF Research Database (Denmark)

    Reid, S.M.; Paton, D.J.; Wilsden, G.

    2004-01-01

    Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus....... The RT-PCR and virus isolation were generally comparable in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization...... of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs....

  2. Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

    Directory of Open Access Journals (Sweden)

    Ruby Chandna

    Full Text Available The real time quantitative reverse transcription PCR (qRT-PCR is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes, ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes, in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

  3. Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler's murine encephalomyelitis virus and rat theilovirus.

    Science.gov (United States)

    Yuan, Wen; Wang, Jing; Xu, Fengjiao; Huang, Bihong; Lian, Yuexiao; Rao, Dan; Yin, Xueqin; Wu, Miaoli; Zhu, Yujun; Zhang, Yu; Huang, Ren; Guo, Pengju

    2016-10-01

    Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation PCR and conventional RT-PCR. For 439 clinical samples,95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses.

  4. 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.

    Science.gov (United States)

    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.

  5. 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.

    Science.gov (United States)

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

    Institute of Scientific and Technical Information of China (English)

    LIAN Hong-xia; LU De-xun; GAO Min

    2009-01-01

    Porcine lipoprotein lipase (LPL) cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP 10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR (FQ-PCR). The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 103 to 1010 copies. The standard curves showed high correlations (R2=0.9871). A series of standards for real-time PCR analysis have been constructed successfully, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.

  7. Expression Levels of RFP in Normal and Cancer Human Tissues via Real-time RT-PCR Detection

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Ret finger protein(RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger of motif, a B-box, and a coiled-coil domain(they are called RBCC generally). Although RFP was known to be an oncogene when its RBCC moiety was connected with a tyrosine kinase domain by DNA rearrangement, its biological function was not well defined. In this study, by using real-time RT-PCR, the RFP expressions in human and mouse normal tissues, and in the cervical squamous cell carcinoma, endometrial adenocarcinoma, gastric adenocarcinoma, esophageal squamous cell carcinoma, and brain cancer tissues were analyzed. The result of the study proved that the highest level of mRNA reverse transcription appeared in the normal testical tissue, whereas that in other normal tissues of human and mice were low. The mRNA reverse transcription level of RFP was higher in the endometrial adenocarcinoma tissue than in the cervical squamous cell carcinoma tissue; the mRNA reverse transcription level of RFP in the gastric adenocarcinoma tissue was significantly higher than that in the esophageal squamous cell carcinoma tissue. It was also found that the mRNA reverse transcription level of RFP in the brain cancer tissue was higher than that in the normal brain tissue. These results suggested that RFP could possibly be a useful molecular target for the development of new therapeutics for malignant tumors.

  8. [Rapid detection of novel avian influenza virus subtype H7N9 by multiplex real-time RT-PCR].

    Science.gov (United States)

    Luo, Bao-Zheng; Mo, Qiu-Hua; Li, Ru-Shu; Bo, Qing-Ru; Xu, Hai-Nie; Sha, Cai-Hua; Liao, Xiu-Yun

    2014-01-01

    In order to develop a rapid detection kit for novel avian influenza virus (AIV) subtype H7N9, two sets of specific primers and probes were designed based on the nucleotide sequences of hemagglutinin antigen (HA) and neuraminidase antigen (NA) of novel H7N9 virus (2013) available in GenBank to establish the method of TaqMan probe-based multiplex real-time RT-PCR for rapid detection of AIV subtype H7N9. The primer and probe of HA were for all H7 subtype AIVs, while the primer and probe of NA were only for novel N9 subtype AIVs. The results showed that this method had high sensitivity and specificity. This method was applicable to the testing of positive standard sample with a minimum concentration of 10 copies/microL; it not only distinguished H7 subtype from H1, H3, H5, H6, and H9 subtypes, but also distinguished novel N9 subtype from traditional N9 subtype. A total of 2700 samples from Zhuhai, China were tested by this method, and the results were as expected. For the advantages of sensitivity and specificity, the method holds promise for wide application.

  9. Development of a real-time RT-PCR assay for the detection of Crimean-Congo hemorrhagic fever virus.

    Science.gov (United States)

    Atkinson, Barry; Chamberlain, John; Logue, Christopher H; Cook, Nicola; Bruce, Christine; Dowall, Stuart D; Hewson, Roger

    2012-09-01

    Crimean-Congo hemorrhagic fever (CCHF) is a virulent tick-borne disease with a case fatality rate ranging from 10-50% for tick-borne transmission, and up to 80% for nosocomial transmission. Human cases have been reported in over 30 countries across Europe, Asia, and Africa. It appears to be spreading to new areas with several countries reporting their first human cases of CCHF disease within the past 10 years. We report a novel real-time RT-PCR assay designed to amplify a conserved region of the CCHF virus S segment. It is capable of detecting strains from all 7 groups of CCHF, including the AP92 strain that until recently represented a lineage of strains that were not associated with human disease. The limit of detection of the assay is 5 copies of target RNA, and the assay shows no cross-reactivity with other viruses from within the same genus, or with viruses causing similar human disease.

  10. Quantitative real-time RT-PCR of CD24 mRNA in the detection of prostate cancer

    Directory of Open Access Journals (Sweden)

    Christoph F

    2006-03-01

    Full Text Available Abstract Background Gene expression profiling has recently shown that the mRNA for CD24 is overexpressed in prostate carcinomas (Pca compared to benign or normal prostate epithelial tissues. Immunohistochemical studies have reported the usefulness of anti-CD24 for detecting prostate cancer over the full range of prostate specimens encountered in surgical pathology, e.g. needle biopsies, transurethral resection of prostate chips, or prostatectomies. It is a small mucin-like cell surface protein and thus promises to become at least a standard adjunctive stain for atypical prostate biopsies. We tested the usefulness of real-time RT-PCR for specific and sensitive detection of CD24 transcripts as a supplementary measure for discriminating between malignant and benign lesions in prostatic tissues. Methods Total RNA was isolated from snap-frozen chips in 55 cases of benign prostatic hyperplasia (BPH and from frozen sections in 59 prostatectomy cases. The latter contain at least 50% malignant epithelia. Relative quantification of CD24 transcripts was performed on the LightCycler instrument using hybridization probes for detection and porphobilinogen deaminase transcripts (PBGD for normalization. Results Normalized CD24 transcript levels showed an average 2.69-fold increase in 59 Pca-cases (mean 0.21 when compared to 55 cases of BPH (mean 0.08. This difference was highly significant (p Conclusion The present study demonstrates the feasibility of quantitative CD24 RNA transcript detection in prostatic tissues even without previous laser microdissection.

  11. Estrogen receptors alpha and beta in rat placenta: detection by RT-PCR, real time PCR and Western blotting

    Directory of Open Access Journals (Sweden)

    Al-Bader Maie D

    2006-03-01

    Full Text Available Abstract Background High levels of estrogens during pregnancy not only retard placental and fetal growth but can lead to reproductive tract abnormalities in male progeny. Estrogens act through estrogen receptors (ER to modulate the transcription of target genes. These ER exist in two isoforms, ER alpha and ER beta and recently several variants of these isoforms have been identified. Methods The expressions of ER isoforms and variants have been studied in rat placenta at 16, 19 and 21 days gestation (dg. Gene expression was assessed using RT-PCR and real time PCR while protein expression was studied using Western blotting followed by immunodetection. Placental homogenates were probed with: a monoclonal antibody raised against the steroid binding domain of the ER alpha (ER alpha -S, a monoclonal antibody raised against the hinge region of ER alpha (ER alpha -H and a polyclonal antibody raised against the amino terminus of ER beta. Results ER alpha and ER beta mRNA and protein were detected from as early as 16 dg. Two PCR products were detected for ER alpha, one for the wild type ER alpha, and a smaller variant. Real time PCR results suggested the presence of a single product for ER beta. The antibodies used for detection of ER alpha protein both identified a single 67 kDa isoform; however a second 54 kDa band, which may be an ER alpha variant, was identified when using the ER alpha -H antibody. The abundance of both ER alpha bands decreased significantly between 16 and 19 dg. As for ER beta, four bands (76, 59, 54 and 41 kDa were detected. The abundance of the 59 and 54 kDa bands decreased significantly between 16 and 19 dg. Conclusion This study shows that both ER protein isoforms and their variants are present in rat placenta. The decrease in their expression near parturition suggests that the placenta may be relatively unresponsive to estrogens at this stage.

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

    Science.gov (United States)

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

    2010-05-18

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

  13. Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; de Stricker, K.;

    2003-01-01

    Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting...

  14. Relative quantification and detection of different types of infectious bursal disease virus in bursa of Fabricius and cloacal swabs using real time RT-PCR SYBR green technology

    DEFF Research Database (Denmark)

    Li, Yiping; Handberg, K.J.; Kabell, Susanne

    2007-01-01

    In present study, different types of infectious bursal disease virus (IBDV), virulent strain DK01, classic strain F52/70 and vaccine strain D78 were quantified and detected in infected bursa of Fabricius (BF) and cloacal swabs using quantitative real time RT-PCR with SYBR green dye. For selection...

  15. Validation of reference genes for gene expression studies in the dinoflagellateAkashiwo sanguinea by quantitative real-time RT-PCR

    Institute of Scientific and Technical Information of China (English)

    DENG Yunyan; HU Zhangxi; MA Zhaopeng; TANG Ying Zhong

    2016-01-01

    The accurate measurement of gene expression via quantitative real-time reverse transcription PCR (qRT-PCR) heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of qRT-PCR to the HAB-forming group. In this study, six candidate reference genes, 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH),α-tubulin (TUA),β-tubulin (TUB), actin (ACT) and cytochrome oxidase subunit 1 (COX1), were evaluated for their expression stability with qRT-PCR and three statistical algorithms (GeNorm, NormFinder, and BestKeeper) for the cosmopolitan, harmful algal bloom-forming dinoflagellateAkashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages (resting cysts), different growth stages, in darkness, exposed to abscisic acid (ABA) and exposed to temperature stress. The results indicated thatTUA,18S andGAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs ofACT andTUA,18S andGAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination ofTUA andTUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair ofACT andCOX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle, particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates

  16. Development and evaluation of a SYBR green-based real time RT-PCR assay for detection of the emerging avian influenza A (H7N9 virus.

    Directory of Open Access Journals (Sweden)

    Zheng Zhu

    Full Text Available Most recently a novel avian-origin influenza A (H7N9 virus emerged in China and has been associated with lots of human infection and fatal cases. Genetic analysis of the viral genome revealed that this reassortant virus might be better adapted to humans than other avian influenza viruses. Molecular diagnostic methods are thus urgently needed in public health laboratories. In this study, a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR was developed to detect the novel H7N9 virus. The primer pairs on the basis of the hemagglutinin and neuraminidase gene sequences of H7N9 viruses amplified subtype-specific fragments with Tm values of 80.77±0.06°C for H7 and 81.20±0.17°C for N9 respectively. The standard curves showed a dynamic linear range across 6 log units of RNA copy number (10(6 to 10(1 copies/ µl with a detection limit of 10 copies per reaction for both H7 and N9 assays by using serial ten-fold diluted in-vitro transcribed viral RNA. In addition, no cross-reactivity was observed with seasonal H1N1, H1N1 pdm09, H3N2, H5N1 and H9N2 viruses as well as other human respiratory viruses. When the assay was further evaluated in H7N9 virus infected clinical samples, positive amplification signals were obtained in all of the specimens with the accordance between H7 and N9 assays. Therefore, the established SYBR green-based real time RT-PCR assay could provide a rapid, sensitive, specific and reliable alternative approach with lower costs for high throughput screening of suspected samples from humans, animals and environments in first line public health laboratories.

  17. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus.

    Directory of Open Access Journals (Sweden)

    Gilberto A Santiago

    Full Text Available Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV. There are four genetically distinct DENVs (DENV-1-4 that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1-4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1-4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1-4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

  18. Sequence Optimized Real-Time RT-PCR Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus

    Science.gov (United States)

    2017-03-21

    density macroarrays [15], high density resequencing arrays [16], 55 padlock probes with colorimetric readout [17], LAMP [18], and polymerase chain reaction ...Announcement). Briefly, the S segment of each virus was amplified using the 83 SuperScript III One-Step RT- PCR system with Platinum Taq DNA Polymerase High...Platinum One-Step Quantitative RT- PCR System) # Rxns = Reagents Stock [Final] 1rxn 28 2X Reaction Mix 2 1 10 280 Nuclease-free water 1.7 47.6 100 µM F

  19. Ekspresi protein vimetin dan neurofilamen pada tunas anggota depan mencit black-6 umur kebuntingan 12 hari akibat induksi 2-metoksietanol secara Real Time RT-PCR

    Directory of Open Access Journals (Sweden)

    YULIA IRNIDAYANTI

    2011-11-01

    Full Text Available Irnidayanti Y. 2011. Ekspresi protein vimetin dan neurofilamen pada tunas anggota depan mencit black-6 umur kebuntingan 12 hari akibat induksi 2-metoksietanol secara Real Time RT-PCR. Bioteknologi 8: 53-58. Tujuan penelitian ini adalah untuk mengetahui dampak dari 2-metoksietanol, bahan kimia utama industri plastik. Analisis ekspresi gen semakin penting dalam penelitian biologi, dimana real-time reverse transcription PCR (RT-PCR menjadi metode yang dipilih untuk mendapatkan profil ekspresi dari seleksi gen secara akurat. Mencit black-6 bunting diinjeksi 2-metoksietanol secara intraperitoneal dengan 7,5 mmol/kg pada umur kebuntingan 100 hari. Embrio diperoleh pada umur kebuntingan 12 hari. Tunas anggota depan embrio dikumpulkan dan dimasukkan ke dalam tabung, yang mengandung larutan RNA-latter. Untuk mengetahui perubahan ekspresi gen tunas anggota depan yang disebabkan oleh induksi 2-metoksietanol digunakaan RT-PCR dalam penelitiaan ini. Dalam percobaan digunakan teknologi RT-PCR Li ght Cycler. Hasil penelitiaan menunjukkan bahwa injeksi 2-metoksietanol, pada periode pralahir terutama pada umur kebuntingan 12 hari, ekspresi vimentin pada tunas anggota depan mencit perlakuan meningkatkan dibandingkan kontrol. Sementara itu ekspresi neurofilamen cenderung menurun, secara tidak langsung tidak disebabkan oleh injeksi 2-metoksietanol.

  20. Measurement of cytokine mRNA expression in intestinal biopsies of cats with inflammatory enteropathy using quantitative real-time RT-PCR.

    Science.gov (United States)

    Nguyen Van, N; Taglinger, K; Helps, C R; Tasker, S; Gruffydd-Jones, T J; Day, M J

    2006-10-15

    Inflammatory bowel disease (IBD) is a common condition in cats characterised by infiltration of inflammatory cells into the intestinal mucosa. In this study, real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantify cytokine messenger RNA (mRNA) expression in intestinal biopsies from cats. Biopsies were collected from seven cats with chronic diarrhoea and histologically confirmed IBD, five cats with chronic diarrhoea due to non-IBD gastrointestinal (GI) disease, and nine clinically normal cats with or without subclinical inflammatory changes in small intestine. Real-time RT-PCR was developed for quantification of mRNA encoding interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p35 and p40), IL-18, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a 'housekeeper' gene. All real-time PCR efficiencies were>90% (range 90.4-102%) with correlation coefficients >0.99 (range 0.998-1). The results of the study were analyzed on the basis of either clinical presentation or histopathological evidence of intestinal inflammation. The former analysis showed that mRNA encoding IL-10 and TGF-beta (immunoregulatory cytokines), and IL-6, IL-18, TNF-alpha and IL-12 p40 (Th1 and pro-inflammatory cytokines) was significantly higher in clinically normal cats and cats with IBD when compared to cats with other GI diseases. IL-5 mRNA was significantly higher in cats with IBD compared to clinically normal cats. IL-2 mRNA was significantly lower in cats with non-IBD GI disease than in clinically normal cats. Analysis on the basis of histopathological change revealed that cats with intestinal inflammation had significantly more transcription of genes encoding IL-6, IL-10, IL-12p40, TNF-alpha and TGF-beta than those with normal intestinal morphology. The results suggest that immune dysregulation plays a role in feline IBD and that IBD

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Ashutosh Wadhwa

    2017-01-01

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

  3. 禽肺炎病毒实时荧光定量 RT-PCR 检测方法的建立%Development of Real-time RT-PCR for Detection of Avian Pneumovirus in Chickens

    Institute of Scientific and Technical Information of China (English)

    谢志勤; 谢芝勋; 刘加波; 庞耀珊; 邓显文; 谢丽基; 范晴; 罗思思

    2014-01-01

    A SYBR Green Ⅰ real-time RT-PCR was developed and applied to detect avian pneumovirus subtype C.A pair of specific primers were designed and synthesized according to the fusion gene of avian pneumovirus subtype C in GenBank.The reaction parameters,such as the concentration of primers,and the reaction buffer,were optimized to develop a SYBR Green Ⅰ real-time RT-PCR for the rapid detection of avian pneumovirus subtype C.The sensitivity and specificity were tested by this method.The results in-dicated that only avian pneumovirus strain had the positive curve in this assay.As limit as 10 copies of plas-mid DNA isolated from competent cells that transferred avian pneumovirus gene was detected in this test. 54 of clinical samples collected from chicken lungs and trachea were detected by this method.It showed that all of clinical samples were negative for avian pneumovirus subtype C.It indicated that this SYBR Green Ⅰ real-time RT-PCR was a good method for detection of avian pneumovirus.It was a quick,sensi-tive,specific and quantitative method for identification of avian pneumovirus in chickens in the future.%根据 C 亚型禽肺炎病毒 F 基因序列,设计了一对针对 C 亚型禽肺炎病毒的特异性引物,应用该引物建立了 SYBR Green Ⅰ实时荧光定量 RT-PCR 检测 C 亚型禽肺炎病毒的方法,并对建立的方法进行特异性、敏感性测定和临床样品检验。经检验该方法能特异性地鉴别检测禽肺炎病毒,特异性好;敏感性可检测到10个拷贝的禽肺炎病毒质粒 DNA 模板;对收集的54份鸡肺病料样品进行检测,没有检测到 C 亚型禽肺炎病毒阳性病料。建立的 SYBR Green Ⅰ实时荧光定量 RT-PCR 方法检测 C 亚型禽肺炎病毒具有特异、敏感、快速、定量等优点,用该方法检测证实,采集的临床样品中还没有 C 亚型禽肺炎病毒的存在。

  4. Selection of Reference Genes for Quantitative Real-Time RT-PCR Studies in Tomato Fruit of the Genotype MT-Rg1

    Science.gov (United States)

    González-Aguilera, Karla L.; Saad, Carolina F.; Chávez Montes, Ricardo A.; Alves-Ferreira, Marcio; de Folter, Stefan

    2016-01-01

    Quantitative real-time RT-PCR (qRT-PCR) has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L.) cultivar Micro-Tom (MT) is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed, and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 and APETALA2c during fruit development are comparable to previous reports using other tomato cultivars. PMID:27679646

  5. A comparative study on the use of real time polymerase chain reaction (RT-PCR and standard isolation techniques for the detection of Salmonellae in broiler c

    Directory of Open Access Journals (Sweden)

    Waleed A. Ibrahim

    2014-06-01

    Full Text Available This study was carried out to compare between conventional cultural isolation methods and real time polymerase chain reaction (RT-PCR technique for the detection of Salmonella in broiler chicks. About 120 livers and intestinal contents samples were collected from 1800 day-old imported and local broiler chicks. The incidence of Salmonellae among imported chicks was 11.67% compared to 21.67% among local chicks using conventional cultural isolation methods. Salmonella newport (S. newport showed the highest incidence rate in imported chicks, while Salmonella enteritidis and Salmonella typhimurium were frequently detected in local chicks. The RT-PCR results for detection of invA gene of Salmonella spp. were 58.33% and 66.67% positive samples in imported and local chicks, respectively. Results have confirmed that RT-PCR technique is rapid, robust, effective and reliable method for detection of Salmonella spp. in broiler chicken when compared to conventional cultural methods. However, RT-PCR should be performed parallel with conventional methods for more accurate detection results of different Salmonellae serovars.

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

    Science.gov (United States)

    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 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.

  7. CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use

    Directory of Open Access Journals (Sweden)

    Mercereau-Puijalon Odile

    2005-03-01

    The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications.

  8. Duplex real-time qRT-PCR for the detection of hepatitis A virus in water and raspberries using the MS2 bacteriophage as a process control.

    Science.gov (United States)

    Blaise-Boisseau, Sandra; Hennechart-Collette, Catherine; Guillier, Laurent; Perelle, Sylvie

    2010-06-01

    Hepatitis A virus (HAV) infection is the leading worldwide cause of acute viral hepatitis. An important aspect of viral control is rapid diagnosis. Epidemiological studies have linked hepatitis A outbreaks to the consumption of drinking water or soft fruits exposed to faecal contamination. Real-time reverse transcriptase PCR (qRT-PCR) is now widely used for detecting RNA viruses in food samples. Efficiency of viral concentration, nucleic acid extraction and the presence of potential inhibitors of the RT-PCR reaction must be monitored to prevent false negative results. In this study, the MS2 bacteriophage used as a process control was detected simultaneously with HAV in a one-step duplex real-time qRT-PCR. The assay was developed for testing water and raspberries. Adding MS2 showed no loss of sensitivity for HAV detection in water and raspberry samples. The limit of detection of HAV with this new approach was 10PFU for 1.5L of bottled water, 100PFU for 1.5L of tap water, 50PFU for 25g of fresh raspberries and 100PFU for 25g of frozen raspberries. The data show that the MS2 offers a very reliable and simple way to monitor false-negative results, making it a valuable tool in the routine diagnostics laboratory.

  9. Rapid diagnosis of acute hemorrhagic conjunctivitis due to coxsackievirus A24 variant by real-time one-step RT-PCR.

    Science.gov (United States)

    Lévêque, Nicolas; Lahlou Amine, Idriss; Tcheng, Remy; Falcon, Delphine; Rivat, Nathalie; Dussart, Philippe; Muyembe, Jean-Jacques; Chomel, Jean-Jacques; Norder, Helene; Eugene, Maxime; Lina, Bruno

    2007-06-01

    Coxsackievirus A24 variant is, together with enterovirus 70 and adenoviruses, the major etiological agent involved in acute hemorrhagic conjunctivitis outbreaks worldwide. However, the standard virus isolation method followed by serotyping or VP1 region sequencing is time-consuming. A rapid method for the detection of coxsackievirus A24 variant from conjunctival swab specimens would be useful in the context of explosive and extensive outbreaks. A one-step real-time RT-PCR assay based on TaqMan technology was thus developed and assessed on 36 conjunctival swabs from outbreaks of conjunctivitis in Morocco in 2004 due to a coxsackievirus A24 variant and in Corsica in 2006 due to adenovirus type 3, and 83 virus strains including 41 coxsackievirus A24 variant collected in French Guiana and Guadeloupe in 2003, in the Democratic Republic of the Congo in 2003, in Morocco in 2004 and 42 other virus species genetically close or known to be responsible for conjunctivitis. All the conjunctival swabs from coxsackievirus A24 variant related outbreak and the 41 coxsackievirus A24 variant strains were tested positive by the RT-PCR assay within 4h. This novel single-tube real-time RT-PCR assay is sensitive and specific, and consists in a reliable and faster alternative to the viral culture for recent and future acute hemorrhagic conjunctivitis outbreaks caused by coxsackievirus A24 variant.

  10. Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa

    DEFF Research Database (Denmark)

    Bachanek-Bankowska, Katarzyna; Mero, Herieth R.; Wadsworth, Jemma

    2016-01-01

    the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce...... methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within...... CT values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide...

  11. Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. II. Diagnostic evaluation of two protocols.

    Science.gov (United States)

    Warg, Janet V; Clement, Travis; Cornwell, Emily R; Cruz, Angela; Getchell, Rodman G; Giray, Cem; Goodwin, Andrew E; Groocock, Geoffrey H; Faisal, Mohamed; Kim, Robert; Merry, Gwenn E; Phelps, Nicholas B D; Reising, Monica M; Standish, Isaac; Zhang, Yan; Toohey-Kurth, Kathy

    2014-08-21

    Two real-time reverse transcription polymerase chain reaction (rRT-PCR) assays under consideration for deployment to multiple testing laboratories across the USA were evaluated for diagnostic sensitivity and specificity on tissue homogenates obtained from natural and experimental viral hemorrhagic septicemia (VHS)-infected fish. Estimates for diagnostic specificity using virus isolation as the reference method were similar between laboratories regardless of the assay. Diagnostic sensitivity estimates of 0.96 (95% CI: 0.95, 0.97) for Jonstrup et al. (2013)'s assay (J Fish Dis 36:9-23) exceeded the diagnostic sensitivity of 0.85 (95% CI: 0.83, 0.87) for Phelps et al. (2012)'s assay (J Aquat Anim Health 24:238-243). The Jonstrup rRT-PCR assay is robust as demonstrated by high sensitivity and specificity estimates across laboratories and can be used as a valuable tool for targeted surveillance and for testing of suspect VHSV samples.

  12. Real-time RT-PCR assay to differentiate clades of H5N1 avian influenza viruses circulating in Vietnam.

    Science.gov (United States)

    Kis, Z; Jones, J; Creanga, A; Ferdinand, K; Inui, K; Gerloff, N; Davis, C T; Nguyen, T; Donis, R O

    2013-11-01

    Continued circulation and geographical expansion of highly pathogenic avian influenza H5N1 virus have led to the emergence of numerous clades in Vietnam. Although viral RNA sequencing and phylogenetic analysis are the gold standard for H5N1 HA clade designation, limited sequencing capacity in many laboratories precludes rapid H5N1 clade identification and detection of novel viruses. Therefore, a Taqman real-time RT-PCR assay for rapid differentiation of the four major H5N1 clades detected in Vietnam was developed. Using HA sequence alignments of clades 1.1, 2.3.2.1, 2.3.4, and 7 viruses, primers and FAM-labeled probes were designed to target conserved regions characteristic of each clade. The assay was optimized and evaluated using circulating clades of H5N1 collected in Vietnam from 2007 to 2012 and shown to be both sensitive and specific for the differentiation of the four H5N1 clades. The assay provides a useful tool for screening of large specimen collections for HA gene sequencing and phylogenetic analysis and for the rapid identification of molecular clade signatures to support outbreak investigations and surveillance activities. Finally, this assay may be useful to monitor for the emergence of novel or variant clades of H5N1 in Vietnam in the future or in other countries where these particular clades may circulate.

  13. Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR for detection of avian metapneumovirus subtype A Comparação entre as técnicas de RT-PCR convencional e RT-PCR em tempo real para a detecção do metapneumovírus aviários subtipo A

    Directory of Open Access Journals (Sweden)

    Helena Lage Ferreira

    2009-08-01

    Full Text Available Avian metapneumovirus (AMPV belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F gene and nucleocapsid (N gene were compared with an established test for the attachment (G gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3 to 10¹ TCID50 mL-1. The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.O metapneumovírus aviário (AMPV pertence ao gênero Metapneumovirus, família Paramyxoviridae. Isolamento viral, sorologia e detecção do RNA genômico são atualmente as técnicas utilizadas para o diagnóstico desse agente. O objetivo do presente estudo foi comparar a detecção de RNA viral de seis isolados de AMPV, subtipo A (AMPV/A, utilizando diferentes métodos de RT-PCR convencional e real time RT-PCR (RRT-PCR. Duas novas técnicas de RT-PCR convencional e duas técnicas de RRT-PCR, ambas para a detecção dos genes da nucleoproteína (N e da proteína de fusão (F, foram comparadas com um RT-PCR previamente estabelecido para a detecção do AMPV (gene da glicoproteína -G. Todos esses métodos foram capazes de detectar os isolados AMPV/A. As técnicas RRT-PCR (genes F e N mostraram os menores limites de detecção (10(0.3 to 10¹ TCID50 mL-1. Os resultados sugerem que as técnicas RT-PCR convencional (gene F e as técnicas de RRT-PCR (gene F e N desenvolvidas no presente estudo podem ser utilizadas com sucesso para a detecção do

  14. Comparative Study on Real-time PCR and RT-PCR Testing Methods for Detecting human Metapneumo Virus%Real-time PCR与RT-PCR检测儿童人偏肺病毒

    Institute of Scientific and Technical Information of China (English)

    郭鑫; 崔玉霞; 诸葛姝芮; 刘兴梅

    2015-01-01

    目的:比较荧光定量PCR( real-time PCR)与普通反转录酶-聚合酶链锁反应( RT-PCR)对人偏肺病毒( hMPV)的检测价值。方法:Real-time PCR和RT-PCR同时对500例急性下呼吸道感染( ALRTI)患儿鼻咽部分泌物进行hMPV检测,分析两种方法的特异性和灵敏性。结果:Real-time PCR和RT-PCR的检出率分别为16%及9.8%,RT-PCR与Real-time PCR比较,灵敏度为31.3%(25/80),特异度为94.3%(396/420)。结论:Real-time PCR检测hMPV敏感性高于RT-PCR,是临床检测儿童鼻咽部分泌物hMPV感染的有效的方法。%Objective:To investigate the effect of real-time quantitative PCR and RT-PCR in detec-tion of human metapneumo virus. Methods:Real-time PCR and RT-PCR are adopted to test hMPV on throat exudate of 500 acute lower respiratory tract infection( ALRTI)child patients,analyzing the spe-cificity and sensitivity of both assays. Results:Comparing Real-time PCR and RT-PCR,the detection rates were 16 % to 9. 8%. Comparing Real-time PCR and RT-PCR,the sensitivity and specificity were 31. 3 %(25/80)and 94. 3%(396/420)respectively. Conclusion:The sensitivity of Real-time PCR is significantly higher than RT-PCR in detection of hMPV,which provid an effective method for detection of hMPV.

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

    Science.gov (United States)

    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 viruses in 112 NPA and 35 (34.3%) and 31 (30.4%) in 102 NPS, respectively (P viruses in CAP adults. Both molecular techniques yielded better results with nasopharyngeal aspirate than swabs.

  16. Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols.

    Science.gov (United States)

    Warg, Janet V; Clement, Travis; Cornwell, Emily R; Cruz, Angela; Getchell, Rodman G; Giray, Cem; Goodwin, Andrew E; Groocock, Geoffrey H; Faisal, Mohamed; Kim, Robert; Merry, Gwenn E; Phelps, Nicholas B D; Reising, Monica M; Standish, Isaac; Zhang, Yan; Toohey-Kurth, Kathy

    2014-08-21

    Eight laboratories worked collectively to evaluate 4 real-time RT-PCR (rRT-PCR) protocols targeting viral hemorrhagic septicemia virus (VHSV) being considered for deployment to a USA laboratory testing network. The protocols utilized previously published primers and probe sets developed for detection and surveillance of VHSV. All participating laboratories received and followed a standard operating protocol for extraction and for each of the rRT-PCR assays. Performance measures specifically evaluated included limit of detection (defined as the smallest amount of analyte in which 95% of the samples are classified as positive), analytical specificity, assay efficiency across genotype representatives, within- and between-plate variation within a laboratory, and variation between laboratories using the same platform, between platforms, and between software versions. This evaluation clearly demonstrated that the TaqMan®-based assay developed by Jonstrup et al. (2013; J Fish Dis 36:9-23) produced the most consistent analytical performance characteristics for detecting all genotypes of VHSV across the 8 participating laboratories.

  17. Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland.

    LENUS (Irish Health Repository)

    Menton, John F

    2007-01-01

    BACKGROUND: Noroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals. RESULTS: A real-time RT-PCR assay and a Reverse Line Blot Hybridisation assay were developed based on the ORF1-ORF2 region. The sensitivity and reactivity of the two assays used was validated using a reference stool panel containing 14 NoV genotypes. The assays were then used to investigate two outbreaks of gastroenteritis in two Irish hospitals. 56 samples were screened for NoV using a real-time RT-PCR assay and 26 samples were found to be positive. Genotyping of these positive samples found that all positives belonged to the GII\\/4 variant of NoV. CONCLUSION: The combination of the Real-time assay and the reverse line blot hybridisation assay provided a fast and accurate method to investigate a NoV associated outbreak. It was concluded that the predominant genotype circulating in these Irish hospitals was GII\\/4 which has been associated with the majority of NoV outbreaks worldwide. The assays developed in this study are useful tools for investigating NoV infection.

  18. Lesch-Nyhan variant syndrome: real-time rt-PCR for mRNA quantification in variable presentation in three affected family members.

    Science.gov (United States)

    Nguyen, Khue Vu; Naviaux, Robert K; Paik, Kacie K; Nakayama, Tomohiro; Nyhan, William L

    2012-01-01

    Inherited mutations of hypoxanthine guanine phosphoribosyltransferase (HPRT) give rise to Lesch-Nyhan syndrome (LNS) or variants (LNV). We report molecular insights from real-time RT-PCR for HPRT mRNA quantification into the mechanism by which a single mutation located in exon 7 of the HPRT gene: c.500G>T, p.R167M, led to different clinical phenotypes from three male LNV-affected patients in the same family manifesting parallel differences in enzymatic activities. This approach can be applied for understanding genotype-phenotype correlations for other human genetic diseases.

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

    Directory of Open Access Journals (Sweden)

    Aman Kamboj

    2014-01-01

    Full Text Available 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.

  20. Screening and detection of human enterovirus 71 infection by a real-time RT-PCR assay in Marseille, France, 2009-2011

    OpenAIRE

    Tan, C. Y. Q.; Gonfrier, G.; Ninove, L.; Zandotti, C.; Dubot Pérès, Audrey; De Lamballerie, Xavier; Charrel, R.N.

    2012-01-01

    Enterovirus-positive samples diagnosed in Marseille (January 2009 to September 2011) were screened for EV71 by real-time RT-PCR. EV71 was detected in three children below the age of 2 years with no history of overseas travel; two of these cases were associated with severe clinical presentation. Viruses demonstrated genetic similarity to other European genogroup C2 strains. Strain MRS/09/3663 complete sequencing revealed 97.6% identity across the entire genome with a 2008 Singapore isolate, wi...

  1. A novel real-time RT-PCR with TaqMan-MGB probes and its application in detecting BVDV infections in dairy farms

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yong-qiang; WANG Zhi-liang; LIU Hai-sheng; WU Xiao-dong; WANG Xiao-zhen; LI Jin-ming; ZHAO Yong-gang; L Yan; REN Wei-jie; GE Sheng-qiang

    2015-01-01

    A real-time RT-PCR assay using TaqMan-MGB probes was developed to detect and type the bovine viral diarrhea virus (BVDV) in cattle. Universal primers and TaqMan-MGB probes were designed from the 5´-untranslated region of known pestiviral sequences. Prior to optimizing the assay, cRNAs were transcribed in vitro from the BVDV 1 and BVDV 2 RT-PCR products to make standard curves. The detection limit of the assay was 1.72×102 copies for BVDV 1 and 2.14×102 copies for BVDV 2. The speciifcity of the assay evaluated on several BVDV strains including bovine herpesvirus 1 (BHV 1), foot and mouth disease virus (FMDV) and several classical swine fever virus (CSFV) strains showed speciifc detection of the positive virus over 40 cycles. The assay was highly reproducible with the coefifcient of variance ranging from 1.04 to 1.33%for BVDV 1 and from 0.83 to 1.48%for BVDV 2, respectively. Using this method, we tested a total of 2 327 cattle from three dairy farms for the presence of BVDV persistently infected (PI) animals. In this assay, each RT-PCR template contained a mixture of ten samples from different animals. The occurrence rate of PI cattle in three farms ranging from 0.9 to 2.54%could represent partly the PI rates in cattle farm in China. In conclusion, using our real-time PCR assay, we could effectively detect and type BVDV and identify PI cattle in a rapid and cost-effective manner.

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

    Directory of Open Access Journals (Sweden)

    Evelyne Picard-Meyer

    2015-01-01

    Full Text Available 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.

  3. Development of a highly sensitive real-time one step RT-PCR combined complementary locked primer technology and conjugated minor groove binder probe

    Directory of Open Access Journals (Sweden)

    Lee Sunhwa

    2011-06-01

    Full Text Available Abstract Background Enterovirus (EV infections are commonly associated with encephalitis and meningitis. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patients, by ruling out other causes of disease. Method To develop a sensitive and reliable assay for routine laboratory diagnosis, we developed a real-time one step reverse transcription polymerase chain reaction (RT-PCR assay with minor groove binder probes and primers modified with complementary locked primer technology (TMC-PCR. We checked the sensitivity of the developed assay by comparing it to a previously published TaqMan probe real-time one-step RT-PCR (TTN-PCR procedure using enteroviral isolates, Enterovirus Proficiency panels from Quality Control on Molecular Diagnostics (QCMD-2007, and clinical specimens from patients with suspected EV infections. Results One hundred clinical specimens from 158 suspected viral meningitis cases were determined to be positive by the TMC-PCR assay (63.29%, whereas only 60 were found to be positive by the TTN-PCR assay (37.97%. The positive and negative agreements between the TMC-PCR and TTN-PCR assays were 100% and 59.2%, respectively. Conclusion This data suggest that the TMC-PCR assay may be suitable for routine diagnostic screening from patient suspected EV infection.

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

    Directory of Open Access Journals (Sweden)

    Shalala eZeynalova

    2015-11-01

    Full Text Available 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 PCR (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 the

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

    Science.gov (United States)

    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

  6. VEGF system expression by immunohistochemistry and real-time RT-PCR study on collared peccary placenta

    DEFF Research Database (Denmark)

    Santos, Tatiana C.; Oliveira, Moacir F.; Papa, Paula C.;

    2014-01-01

    in the placenta and uterus of the collared peccary in nonpregnant females in the luteal phase and throughout pregnancy (>35, 75, 115, and 135 days). The material was examined by immunohistochemistry and by real-time reverse transcription polymerase chain reaction. Intense positive immunolabeling was observed...... in late pregnancy. In the collared peccary, the expression of the VEGF-ligand receptor system was similar to that in porcine and ruminant placentas, suggesting that an epitheliochorial placenta has the same physiological and interhemal barrier during vascular gestational development. The expression...

  7. Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.

    Directory of Open Access Journals (Sweden)

    Zheng Pang

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

  8. The application of SYBR Green I real-time quantitative RT-PCR in quantitative analysis of sweet cherry viruses in different tissues%SYBR Green I实时定量RT-PCR技术在甜樱桃病毒定量分析中的应用

    Institute of Scientific and Technical Information of China (English)

    宗晓娟; 王文文; 王甲威; 魏海蓉; 严霉瑞; 刘庆忠

    2012-01-01

    为了探讨SYBRGreenI实时定量RT—PCR技术在甜樱桃病毒粒子定量分析中的应用前景,以复合感染李属坏死环斑病毒(Prunusnecrotic ringspot virus,PNRSV)、李矮缩病毒(Prune Dwarf vi—rus,PDV)、樱桃病毒A(CherryvirusA,CVA)、樱桃小果病毒一2(Little cherry virus一2,LChV-2)的甜樱桃“红灯”PrunusaviumCV.RedLamp植株为研究对象,采用相对定量方法,分析各病毒的外壳蛋白基因的表达,用以指示病毒的增殖量。在花、幼叶、功能叶、衰老叶中均能检测到4个基因,但各基因表达量在各器官中存在差异。PNRSV-CP与CVA—CP表达模式相似,功能叶中明显高于其它器官,衰老叶中急剧降低。PDV-CP与LChV2一卯表达模式类似,幼叶中的表达量较高,功能叶片中较低。PNRSV-CP在花、功能叶中的表达显著高于其它3个病毒基因。LChV2一cP在各器官中的表达量均低于其余3个基因。该方法适用于植物组织内多种甜樱桃病毒增殖量的分析。%To explain the application of SYBR Green I real-time quantitative RT-PCR in the analysis of the viruses amounts, the plant samples were collected from the sweet cherry trees Prunus avium cv. Red Lamp which were infected by Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Cherry virus A (CVA) and Little cherry virus-2 (LChV-2) simultaneously. Relative expression of the virus coat protein gene was determined and selected to estimate the amounts of the virus in different plant tissues. The results showed that all of the four virus genes can be detected in flowers, young leaves, mature leaves and senescent leaves, but the expression levels of the genes among the samples were different. PNRSV- CP and CVA-CP obtained similar expression patterns, which were high in the functionally active plant tis- sues and low in the senescent tissues. The expression patterns of PDV-CP were similar to that of LChV2- CP, which

  9. Detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time rt-PCR.

    Directory of Open Access Journals (Sweden)

    Kerstin Wernike

    Full Text Available Porcine reproductive and respiratory syndrome (PRRS causes economic losses in the pig industry worldwide, and PRRS viruses (PRRSV are classified into the two distinct genotypes "North American (NA, type 2" and "European (EU, type 1". In 2006, a highly pathogenic NA strain of PRRSV (HP-PRRSV, characterized by high fever as well as high morbidity and mortality, emerged in swine farms in China. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR assay specific for HP-PRRSV was developed and combined with type 1- and type 2-specific RT-qPCR systems. Furthermore, an internal control, based on a heterologous RNA, was successfully introduced. This final multiplex PRRSV RT-qPCR, detecting and typing PRRSV, had an analytical sensitivity of less than 200 copies per µl for the type 1-assay and 20 copies per µl for the type 2- and HP assays and a high diagnostic sensitivity. A panel of reference strains and field isolates was reliably detected and samples from an animal trial with a Chinese HP-PRRS strain were used for test validation. The new multiplex PRRSV RT-qPCR system allows for the first time the highly sensitive detection and rapid differentiation of PRRSV of both genotypes as well as the direct detection of HP-PRRSV.

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

    Science.gov (United States)

    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.

  11. Comparison of two real-time RT-PCR assays for differentiation of C-strain vaccinated from classical swine fever infected pigs and wild boars.

    Science.gov (United States)

    Widén, F; Everett, H; Blome, S; Fernandez Pinero, J; Uttenthal, A; Cortey, M; von Rosen, T; Tignon, M; Liu, L

    2014-10-01

    Classical swine fever is one of the most important infectious diseases for the pig industry worldwide due to its economic impact. Vaccination is an effective means to control disease, however within the EU its regular use is banned owing to the inability to differentiate infected and vaccinated animals, the so called DIVA principle. This inability complicates monitoring of disease and stops international trade thereby limiting use of the vaccine in many regions. The C-strain vaccine is safe to use and gives good protection. It is licensed for emergency vaccination in the EU in event of an outbreak. Two genetic assays that can distinguish between wild type virus and C-strain vaccines have recently been developed. Here the results from a comparison of these two real-time RT-PCR assays in an interlaboratory exercise are presented. Both assays showed similar performance.

  12. Perioperative cancer cell dissemination detected with a real-time RT-PCR assay for EpCAM is not associated with worse prognosis in pancreatic ductal adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Houtmeyers François

    2011-01-01

    Full Text Available Abstract Background Epithelial cell adhesion molecule (EpCAM has been used as surrogate marker for the quantification of circulating tumour cells (CTC. Our aim was to prospectively study the value of a real-time RT-PCR assay for EpCAM detection in the peripheral blood and peritoneal cavity of patients undergoing pancreatectomy for pancreatic ductal adenocarcinoma (PDAC. Methods From 48 patients with PDAC (40 resectable, 8 unresectable and 10 patients with chronic pancreatitis undergoing pancreatectomy 10 ml of venous blood was drawn preoperatively (PB and postoperatively (POB, day 1 (D1B, day 7 (D7B and after 6 weeks (6WB. Of all patients undergoing pancreatectomy, 40 ml peritoneal lavage fluid was taken preoperatively and postoperatively. A real-time RT-PCR assay (TaqMan, ABI Prism 7700 was developed for the detection of EpCAM mRNA. To discriminate between EpCAM-positive and negative samples a cut-off was applied. Median postoperative follow-up was 24.0 months (range: 0.7 - 41.3. Results PB was EpCAM-positive (+ in 25% of patients versus 65% of patients in POB (p At none of the time-points, an association was found between EpCAM positivity in blood and/or peritoneal cavity and cancer-specific or disease-free survival. Also, no significant associations were found between clinicopathological variables and perioperative EpCAM positivity. Conclusions Despite a significant increase in EpCAM counts in postoperative blood and peritoneal lavage fluid this was not associated with worse prognosis after pancreatectomy for PDAC. Trial registration Clinicaltrials.gov NCT00495924

  13. The effect of various disinfectants on detection of avian influenza virus by real time RT-PCR.

    Science.gov (United States)

    Suarez, D L; Spackman, E; Senne, D A; Bulaga, L; Welsch, A C; Froberg, K

    2003-01-01

    An avian influenza (AI) real time reverse transcriptase-polymerase chain reaction (RRT-PCR) test was previously shown to be a rapid and sensitive method to identify AI virus-infected birds in live-bird markets (LBMs). The test can also be used to identify avian influenza virus (AIV) from environmental samples. Consequently, the use of RRT-PCR was being considered as a component of the influenza eradication program in the LBMs to assure that each market was properly cleaned and disinfected before allowing the markets to be restocked. However, the RRT-PCR test cannot differentiate between live and inactivated virus, particularly in environmental samples where the RRT-PCR test potentially could amplify virus that had been inactivated by commonly used disinfectants, resulting in a false positive test result. To determine whether this is a valid concern, a study was conducted in three New Jersey LBMs that were previously shown to be positive for the H7N2 AIV. Environmental samples were collected from all three markets following thorough cleaning and disinfection with a phenolic disinfectant. Influenza virus RNA was detected in at least one environmental sample from two of the three markets when tested by RRT-PCR; however, all samples were negative by virus isolation using the standard egg inoculation procedure. As a result of these findings, laboratory experiments were designed to evaluate several commonly used disinfectants for their ability to inactivate influenza as well as disrupt the RNA so that it could not be detected by the RRT-PCR test. Five disinfectants were tested: phenolic disinfectants (Tek-trol and one-stroke environ), a quaternary ammonia compound (Lysol no-rinse sanitizer), a peroxygen compound (Virkon-S), and sodium hypochlorite (household bleach). All five disinfectants were effective at inactivating AIV at the recommended concentrations, but AIV RNA in samples inactivated with phenolic and quaternary ammonia compounds could still be detected by RRT

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

    Directory of Open Access Journals (Sweden)

    Damodar Paudel

    2011-01-01

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

  15. Development of a Quantitative Real-Time RT-PCR Assay for the Detection of MAGE-A3-Positive Tumors.

    Science.gov (United States)

    Gruselle, Olivier; Coche, Thierry; Louahed, Jamila

    2015-07-01

    Melanoma antigen A3 (MAGE-A3) is a member of the MAGE family of tumor antigens and a relevant candidate for use in cancer immunotherapy. However, not all tumors express MAGE-A3, and closely related members of the MAGE family can be co-expressed with MAGE-A3 in the same tumor. Therefore, in the frame of MAGE-A3 clinical trials, it appeared necessary to evaluate tumors for MAGE-A3 expression with a highly specific quantitative assay to select patients who are eligible for anti-MAGE-A3 immunotherapy treatment. Herein, we describe the development and validation of a quantitative real-time RT-PCR (RT-qPCR) assay for the determination of MAGEA3 gene expression in tumor tissues. In the early phases of development, the designed primers and probe were not able to distinguish between MAGE-A3 and MAGE-A6. To ensure the specificity for MAGE-A3 over MAGE-A6, our strategy was to use a 5'-nuclease probe (or hydrolysis probe). The final assay was shown to be specific and linear within the analytical range, with an acceptable CV for repeatability and intermediate precision. When compared with a reference semiquantitative RT-PCR assay, the two methods were in good agreement, with only 4.23% of the samples giving discordant results. In conclusion, we have developed a MAGE-A3-specific RT-qPCR assay, compatible with a high-throughput setting for the estimation of MAGEA3 gene expression in present and future clinical trials.

  16. Development and validation of a multiplex, real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses.

    Science.gov (United States)

    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.

  17. Development and validation of a multiplex, real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses.

    Directory of Open Access Journals (Sweden)

    Felicity J Haines

    Full Text Available 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.

  18. Estudio comparativo entre una prueba rápida y RT-PCR tiempo real en el diagnóstico de influenza AH1N1 2009 Comparative study of a rapid testing with real time RT-PCR for diagnosis of influenza AH1N1 2009

    Directory of Open Access Journals (Sweden)

    Luz Araceli Castro-Cárdenas

    2011-08-01

    Full Text Available OBJETIVO: Comparar la prueba QuickVue Influenza A+B empleando como estándar la RT-PCR tiempo real para influenza AH1N1 2009. MATERIAL Y MÉTODOS: Estudio retrospectivo-comparativo de 135 muestras de vías respiratorias de individuos sintomáticos para influenza procesadas de mayo 2009 a octubre 2010.Las pruebas citadas se realizaron simultáneamente. Se utilizó el software Confidence Interval Analysis 2000. RESULTADOS: Sensibilidad 62.96; especificidad 94.44; valor predictivo negativo 62.9; valor predictivo positivo 94.44; razón de probabilidad positiva 11.33 y razón de probabilidad negativa 0.39. Se calcularon intervalos de confianza a 95. DISCUSIÓN: Los valores obtenidos concuerdan con otros estudios donde la sensibilidad fluctúa de 50 a 70 y especificidad entre 90 y 95 por ciento. La prueba QuickVue Influenza A+B es rápida, simple y de menor costo que el RT-PCR tiempo real, útil para identificar el tipo de virus en brotes de influenza de una población determinadaOBJECTIVE: Compare QuickVue Influenza A+B test with real-time RT-PCR for the diagnosis of influenza AH1N1 2009. MATERIAL AND METHODS: Retrospective-comparative study of 135 respiratory specimens from individuals with symptoms of influenza processed from May 2009 to October 2010.The above mentioned tests were performed simultaneously. For statistic analysisthe softwareof Confidence IntervalAnalysis 2000 was used. RESULTS: The parameters obtained were: sensitivity 62.96; specificity 94.44; negative predictive value 62.9; positive predictive value 94.44; positive likelihood ratio 11.33; negative likelihood ratio 0.39. Confidence intervals to 95,were calculated to all of the above data. DISCUSSION: The test QuickVue InfluenzaA+B is a rapid,simple test,with lower cost than real-time RT-PCR useful for identifying the type of virus outbreaks of influenza in a given population.It correlates well with more specific test and similar reports.

  19. Characterization of human coronavirus etiology in Chinese adults with acute upper respiratory tract infection by real-time RT-PCR assays.

    Directory of Open Access Journals (Sweden)

    Roujian Lu

    Full Text Available BACKGROUND: In addition to SARS associated coronaviruses, 4 non-SARS related human coronaviruses (HCoVs are recognized as common respiratory pathogens. The etiology and clinical impact of HCoVs in Chinese adults with acute upper respiratory tract infection (URTI needs to be characterized systematically by molecular detection with excellent sensitivity. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we detected 4 non-SARS related HCoV species by real-time RT-PCR in 981 nasopharyngeal swabs collected from March 2009 to February 2011. All specimens were also tested for the presence of other common respiratory viruses and newly identified viruses, human metapneumovirus (hMPV and human bocavirus (HBoV. 157 of the 981 (16.0% nasopharyngeal swabs were positive for HCoVs. The species detected were 229E (96 cases, 9.8%, OC43 (42 cases, 4.3%, HKU1 (16 cases, 1.6% and NL63 (11 cases, 1.1%. HCoV-229E was circulated in 21 of the 24 months of surveillance. The detection rates for both OC43 and NL63 were showed significantly year-to-year variation between 2009/10 and 2010/11, respectively (P<0.001 and P = 0.003, and there was a higher detection frequency of HKU1 in patients aged over 60 years (P = 0.03. 48 of 157(30.57% HCoV positive patients were co-infected. Undifferentiated human rhinoviruses and influenza (Flu A were the most common viruses detected (more than 35% in HCoV co-infections. Respiratory syncytial virus (RSV, human parainfluenza virus (PIV and HBoV were detected in very low rate (less than 1% among adult patients with URTI. CONCLUSIONS/SIGNIFICANCE: All 4 non-SARS-associated HCoVs were more frequently detected by real-time RT-PCR assay in adults with URTI in Beijing and HCoV-229E led to the most prevalent infection. Our study also suggested that all non-SARS-associated HCoVs contribute significantly to URTI in adult patients in China.

  20. 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.

    Science.gov (United States)

    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.

  1. Incidence in diverse pig populations of an IGF2 mutation with potential influence on meat quality and quantity: An assay based on real time PCR (RT-PCR).

    Science.gov (United States)

    Carrodeguas, José Alberto; Burgos, Carmen; Moreno, Carlos; Sánchez, Ana Cristina; Ventanas, Sonia; Tarrafeta, Luis; Barcelona, José Antonio; López, Maria Otilia; Oria, Rosa; López-Buesa, Pascual

    2005-11-01

    IGF2, insulin-like growth factor 2, is implicated in myogenesis and lean meat content. A mutation in a single base (A for G substitution) of the gene for IGF2 (position 3072 in intron 3) has been recently described as the cause of a major QTL effect on muscle growth in pigs [Van Laere, A. S, Nguyen, M., Braunschweig, M., Nezer, C., Collete, C., & Moreau, L. et al. (2003). Nature, 425, 832-836]. We describe here a rapid assay based on real time PCR (RT-PCR) to detect this mutation. We have evaluated the incidence of the mutation in commercial pig crosses, in three populations of purebred Iberian or Iberian×Duroc crosses, and in cured meat products and wild boars. The incidence of the mutation varies among these groups. Penetrance of the A mutation is about 80% in the commercial population. Purebred Iberian pigs were all homozygous G/G whereas crosses of Iberian pigs were heterozygous (90%) or homozygous A/A (10%). The implications of this gene for the selection of Iberian pigs are discussed.

  2. The quantification of tomato microRNAs response to viral infection by stem-loop real-time RT-PCR.

    Science.gov (United States)

    Feng, Junli; Wang, Kai; Liu, Xin; Chen, Shaoning; Chen, Jishuang

    2009-05-15

    MicroRNAs (miRNAs) are RNA molecules consisting of 20-24 nucleotides that play important roles in regulating plant's gene expression for growth and development, cell viability and stress responses. Viral infection often has a noticeable influence on host gene expression, which may result in a range of developmental abnormalities. To investigate the molecular mechanisms underlying viral infection, miRNA pathway and host gene expression, we report herein the application of the novel miRNAs quantification method in tomato, using a stem-loop reverse transcription followed by SYBR Green PCR assay. For the seven tested miRNAs of Solanum lycopersicum, which are related to the regulation of plant development, hormone response, and their own biogenesis, this quantification method showed high sensitivity, specificity, and wide dynamic range. Precise quantification could be achieved with as little as 0.01 ng of total RNAs for most cases. Additionally, their target mRNAs could be quantified from the same RNA sample simultaneously, by the conventional real-time RT-PCR assay. In comparison with mock inoculation, accumulation levels of the tested miRNAs and target mRNAs were found obviously altered in tomato seedlings, indicating that the miRNA pathway was interrupted by Cucumber mosaic virus and Tomato aspermy virus infection.

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

    Science.gov (United States)

    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. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  4. In-house HIV-1 RNA real-time RT-PCR assays: principle, available tests and usefulness in developing countries.

    Science.gov (United States)

    Rouet, François; Ménan, Hervé; Viljoen, Johannes; Ngo-Giang-Huong, Nicole; Mandaliya, Kishor; Valéa, Diane; Lien, Truong Xuan; Danaviah, Sivapragashini; Rousset, Dominique; Ganon, Amandine; Nerrienet, Eric

    2008-09-01

    The principle of currently available licensed HIV-1 RNA assays is based on real-time technologies that continuously monitor the fluorescence emitted by the amplification products. Besides these assays, in-house quantitative (q) real-time reverse transcription (RT)-PCR (RT-qPCR) tests have been developed and evaluated particularly in developing countries, for two main reasons. First, affordable and generalized access to HIV-1 RNA viral load is urgently needed in the context of expected universal access to prevention and antiretroviral treatment programs in these settings. Second, since many non-B subtypes, circulating recombinant forms and unique recombinant forms circulate in these areas, in-house HIV-1 RNA RT-qPCR assays are ideal academic tools to thoroughly evaluate the impact of HIV-1 genetic diversity on the accuracy of HIV-1 RNA quantification, as compared with licensed techniques. To date, at least 15 distinct in-house assays have been designed. They differ by their chemistry and the HIV-1 target sequence (located in gag, Pol-IN or LTR gene). Analytical performances of the tests that have been extensively evaluated appear at least as good as (or even better than) those of approved assays, with regard to HIV-1 strain diversity. Their clinical usefulness has been clearly demonstrated for early diagnosis of pediatric HIV-1 infection and monitoring of highly active antiretroviral therapy efficacy. The LTR-based HIV-1 RNA RT-qPCR assay has been evaluated by several groups under the auspices of the Agence Nationale de Recherches sur le SIDA et les hépatites virales B et C. It exists now as a complete standardized commercial test.

  5. Development and validation of a real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay for investigation of wild poliovirus type 1-South Asian (SOAS) strain reintroduced into Israel, 2013 to 2014.

    Science.gov (United States)

    Hindiyeh, M Y; Moran-Gilad, J; Manor, Y; Ram, D; Shulman, L M; Sofer, D; Mendelson, E

    2014-02-20

    In February 2013, wild poliovirus type 1 (WPV1) was reintroduced into southern Israel and resulted in continuous silent circulation in the highly immune population. As a part of the public health emergency response, a novel real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed, to allow for the sensitive and specific detection of the circulatingWPV1-South Asian (SOAS) strain. Specific primers and probes derived from the VP-1 region were designed, based on sequenced sewage isolates, and used to simultaneously amplify this WPV1-SOAS sequence together with bacteriophage MS-2 as internal control. High titre WPV1-SOAS stock virus was used for assay optimisation and 50 processed sewage samples collected from southern Israel and tested by reference culture based methods were used for analytical validation of the assay’s performance. The limit of detection of the multiplex qRT-PCR (SOAS/MS-2) assay was 0.1 plaque-forming unit (pfu)/reaction (20 pfu/mL) for WPV1-SOAS RNA with 100% sensitivity, specificity, positive and negative predictive values when compared to the culture based method. The turnaround time was rapid, providing results for environmental samples within 24 to 48 hours from completion of sewage processing, instead of five to seven days by culture-based analysis. Direct sewage testing by qRT-PCR assay proved to be a useful tool for rapid detection and environmental surveillance of WPV1-SOAS circulating strain during emergency response. Application of the approach for detection of WPV1-SOAS in stool samples obtained during acute flaccid paralysis (AFP) surveillance or field surveys should be further evaluated.

  6. Detection of West Nile virus by real-time RT-PCR%实时荧光PCR检测西尼罗病毒方法的建立

    Institute of Scientific and Technical Information of China (English)

    邱璐; 李小林; 蒋静; 王艳; 周小俊

    2012-01-01

    西尼罗病毒可引起人畜共患虫媒病,已经在全球许多国家流行.主要随迁飞的鸟类携带扩散,传入我国的风险很大.本研究在参考多株西尼罗病毒基因序列的基础上,设计引物及探针,建立了实时荧光RT-PCR的方法.特异性试验表明,该方法对同为黄病毒科的日本脑炎病毒(JEV) SA14-14-2株和登革病毒(DEN)Ⅰ型cDNA检测均为阴性;敏感性试验证实,实时RT-PCR检测敏感度要比常规RT-PCR法高100倍.说明本研究建立的实时RT-PCR方法敏感性和特异性均较高,可用于西尼罗病毒感染的早期监测和诊断.%West Nile virus ( WNV) is by far the most widely distributed arbovirus. The WNV infection has been recognized as an emerging infectious disease with possible transmitting risk in our country. The pairs of primers and TaqMan probes were designed and synthesized based on the nucleotide sequences of WNV in GenBank. After optimization, the real-time PCR was established. These primers were applied to detect the nucleic acids of WNV, Japanese B encephalitis virus (JEV) and Dengue viruses (DEN) , and the latter two viruses could not be detected. Compared with conventional RT-PCR, real-time PCR appeared to be approximately 100 times more sensitive in detection of WNV. It suggests that the real-time PCR developed for the detection of WNV shows high degree of specificity and sensitivity, and can be used for WNV inspection and investigation.

  7. 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.

    Directory of Open Access Journals (Sweden)

    Nick De Regge

    Full Text Available 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.

  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.

    Science.gov (United States)

    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.

  9. A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes

    NARCIS (Netherlands)

    Thanh, T.T.; Pawestri, H.A.; Ngoc, N.M.; Hien, V.M.; Syahrial, H.; Trung, N.V.; van Doorn, R.H.; Wertheim, H.F.L.; Chau, N.V.V.; Ha, D.Q.; Farrar, J.J.; Hien, T.T.; Sedyaningsih, E.R.; de Jong, M.D.

    2010-01-01

    ABSTRACT: BACKGROUND: The emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants. RESULTS: We developed a single real-time RT-PCR assay for detection of both clades of

  10. 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

    Science.gov (United States)

    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...

  11. 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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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, pmRNA 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.

  13. Effect of blasting treatment and Fn coating on MG63 adhesion and differentiation on titanium: a gene expression study using real-time RT-PCR.

    Science.gov (United States)

    Pegueroles, M; Aguirre, A; Engel, E; Pavon, G; Gil, F J; Planell, J A; Migonney, V; Aparicio, C

    2011-03-01

    Biomaterial surface properties, via alterations in the adsorbed protein layer, and the presence of specific functional groups can influence integrin binding specificity, thereby modulating cell adhesion and differentiation processes. The adsorption of fibronectin, a protein directly involved in osteoblast adhesion to the extracellular matrix, has been related to different physical and chemical properties of biomaterial surfaces. This study used blasting particles of different sizes and chemical compositions to evaluate the response of MG63 osteoblast-like cells on smooth and blasted titanium surfaces, with and without fibronectin coatings, by means of real-time reverse transcription-polymerase chain reaction (qRT-PCR) assays. This response included (a) expression of the α(5), α(v) and α(3) integrin subunits, which can bind to fibronectin through the RGD binding site, and (b) expression of alkaline phosphatase (ALP) and osteocalcin (OC) as cell-differentiation markers. ALP activity and synthesis of OC were also tested. Cells on SiC-blasted Ti surfaces expressed higher amounts of the α(5) mRNA gene than cells on Al(2)O(3)-blasted Ti surfaces. This may be related to the fact that SiC-blasted surfaces adsorbed higher amounts of fibronectin due to their higher surface free energy and therefore provided a higher number of specific cell-binding sites. Fn-coated Ti surfaces decreased α(5) mRNA gene expression, by favoring the formation of other integrins involved in adhesion over α(5)β(1). The changes in α(5) mRNA expression induced by the presence of fibronectin coatings may moreover influence the osteoblast differentiation pathway, as fibronectin coatings on Ti surfaces also decreased both ALP mRNA expression and ALP activity after 14 and 21 days of cell culture.

  14. Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator.

    Science.gov (United States)

    Otis, Jessica P; Ackermann, Laynez W; Denning, Gerene M; Carey, Hannah V

    2010-04-01

    Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.

  15. Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

    Directory of Open Access Journals (Sweden)

    Mary McMillan

    Full Text Available Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA is sufficient for effective normalisation of qRT-PCR data.

  16. Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

    Science.gov (United States)

    McMillan, Mary; Pereg, Lily

    2014-01-01

    Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

  17. The expression by real-time fluorescence quantitive RT-PCR and clinical significance of miR-335 in breast tissue and cancer%实时荧光定量 RT-PCR 检测乳腺组织中 miR-335的表达水平与临床研究

    Institute of Scientific and Technical Information of China (English)

    崔明; 肖苠耀; 何越昆; 乔丹

    2016-01-01

    Objective To detect the expressions of miR-335 in breast tissue and cancer by real-time fluorescence quantitive RT-PCR and investigate the significance of miR-335 in early diagnosis of breast cancer. Methods ABI-7900HT, real time fluorescence quantitative instrument, was used for quantitave analysis of miR-335 in 20 cases of breast cancer tissues and adjacent normal tissues by pathological diagnosis. Results Compared with adjacent normai tissues, the miR-335 in breast cancer tissues showed significant down-regulation(P<0.0001). The relationship of the pregnancy times and the expression lever of miR-335 in normal tissue samples points that: the more times of pregnancy, the higher miR-335 expression in normal tissue (P=0.0371; correlation coefficient=0.59242). Conclusion Breast cancer has obvious alteration in the expression patterns of miR-335, and real time fluorescent quantitative RT-PCR method for detection of miRNA-335 levels in the early diagnosis and treatment of breast cancer provides a new idea and has broad application prospects.%目的:采用实时荧光定量 RT-PCR 分析 miR-335在乳腺癌及癌旁正常组织中的表达水平,探讨 miR-335在乳腺癌疾病中的临床诊断意义。方法运用 ABI-7900HT 实时荧光定量仪对20例已由病理确诊的乳腺癌组织及癌旁正常组织的 miR-335进行了定量分析。结果将乳腺癌与正常组织表达量相比较,发现了 miR-335在所有被检测的乳腺癌组织中均有不同程度的表达水平下调,且结果有显著差异(P<0.0001)。正常组织中的 miR-335表达水平与怀孕次数之间的关系提示:怀孕次数越多,正常组织中的 miR-335表达越高(P=0.0371;相关系数=0.59242)。结论乳腺癌组织及癌旁正常乳腺组织中存在上述 miR-335表达水平的差别,实时荧光定量 RT-PCR 方法检测miRNA-335水平在乳腺癌早期诊断和治疗方面提供了新的思路并具有广阔的应用前景。

  18. 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.

    Science.gov (United States)

    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

  19. Application of reference gene for real-time RT-PCR%内参照基因在实时荧光定量RT-PCR检测中的应用

    Institute of Scientific and Technical Information of China (English)

    陈瑾歆; 陈建业; 李云祥

    2011-01-01

    目的 建立测定内参照β-actin表达量的实时荧光定量RT-PCR两步法检测方法.方法 根据Genbank 中人β-actin保守区域序列设计荧光PCR适用的引物和探针,构建质粒标准品建立标准曲线用于荧光PCR相对定量,检测荧光PCR方法的特异性和重复性.结果 建立了人β-actin实时荧光RT-PCR检测方法.结论 本实验建立的人β-actin表达实时荧光RT-PCR两步法检测方法特异性和重复性较好,为β-actin作为定量RT-PCR中内参照基因进行人其他功能基因和病原基因表达的定量分析奠定了基础.%Objective To establish a Taqman real-time RT-PCR 2-step assay for quantitative detection of the expression of human β-actin. Methods Specific primers and probes were designed for real-time RT-PCR according to β-actin cDNA sequence. Plasmid standard preparations were constructed by T-A clone, extracted and prepared for establishing standard curve used for relative quantification real-time RT-PCR. The expression levels of β-actin were measured by real-time RT-PCR in HepG2 cells to detect the specificity and reproducibility of real-time RT-PCR assay. Results An effective real-time RT-PCR assay was established for detecting β-actin mRNA expression levels.Conclusions The real-time PCR assay for the expression of β-actin is a sensitive, specific tool for quantitative assay of mRNA expression levels of other gene when using β-actin as reference genes.

  20. 联合应用实时量RT-PCR和激光显微切割检测单个肝细胞RNA的表达%Quantitative analysis of RNA levels from single hepatocytes in vivo: combined use of real- time RT- PCR and laser microdissection

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed. To better understand cellular functions, the isolation of single cells and subsequent quantification of the expressed genes is essential. METHODS: Normal liver tissue was obtained from operation, snapfrozen in liquid nitrogen and sectioned in crystat. Individual hepatocytes were microdissected. RNA was extracted, then reverse transcribed and amplified using real- time quantitative polymerase chain reaction (PCR). RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide. In this way,cells were collected, RNA was extracted, reverse transcribed to cDNA and used for analysis of RNA expression by realtime quantitative PCR. The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells. CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real -time PCR. These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.

  1. Real-time onestep RT-PCR for the detection and differentiation of European and North American types of PRRSV in boar semen

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Larsen, Lars Erik

    ) and the virus can be transmitted by this route, creating a need for diagnostic tests to ensure a PRRSV-free semen supply. PCR is an obvious method for such testing, and especially nested and TwoStep RT-PCR methods have been extensively used for this purpose. However, OneStep RT-PCR offers a more convenient......Porcine Reproductive and respiratory syndrome virus (PRRRSV) is a single-stranded RNA virus and a worldwide cause of significant respiratory disease and reproductive failure in swine. Two different types of PRRSV, the European (EU) and North American (US) type exist. Boar semen can harbor PRRSV (1...

  2. Real-time onestep RT-PCR for the detection and differentiation of European and North American types of PRRSV in boar semen

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Larsen, Lars Erik

    Porcine Reproductive and respiratory syndrome virus (PRRRSV) is a single-stranded RNA virus and a worldwide cause of significant respiratory disease and reproductive failure in swine. Two different types of PRRSV, the European (EU) and North American (US) type exist. Boar semen can harbor PRRSV (1......) and the virus can be transmitted by this route, creating a need for diagnostic tests to ensure a PRRSV-free semen supply. PCR is an obvious method for such testing, and especially nested and TwoStep RT-PCR methods have been extensively used for this purpose. However, OneStep RT-PCR offers a more convenient...

  3. Emprego da RT-PCR em tempo real para a quantificação da expressão de genes associados à resposta imune em bezerros bovinos experimentalmente infectados por Neospora caninum Use of the real time RT-PCR for immune related gene expression quantitation in experimentally infected Neospora caninum bovine calves

    Directory of Open Access Journals (Sweden)

    Sandra Mayumi Nishi

    2009-03-01

    samples were analyzed. Real-time RT-PCR detected gene expression in all tissues. No significant variation of GAPDH gene expression was detected among groups, its amplification efficiency was similar to the other genes tested and it was used as the endogenous control for the analysis. Comparisons between infected and uninfected groups allowed the relative gene expression quantification. IFN-γ and TNF-α genes showed increased expression in some samples. iNOS and TGF-β1 genes had some non-significant variations and IL-4 and IL-10 stayed pratically inaltered.

  4. 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.

    Science.gov (United States)

    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.

  5. A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

    Institute of Scientific and Technical Information of China (English)

    Vinayagamurthy Balamurugan; Arnab Sen; Gnanavel Venkatesan; Vinita Yadav; Vandna Bhanot; Veerakyathappa Bhanuprakash; Raj Kumar Singh

    2012-01-01

    A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit(R) for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR.The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50.The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg.Based on the serial dilution of the live-attenuated PPR vaccine virus,the detection limit was ~0.0001 cell culture infectious dose 50% units (TCID50).Additionally,swab materials spiked with known titre of vaccine virus were equally well detected in the assay.The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples.The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples.The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats.Therefore,the established,simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

  6. The use of a one-step real-time reverse transcription polymerase chain reaction (rRT-PCR) for the surveillance of viral hemorrhagic septicemia virus (VHSV) in Minnesota.

    Science.gov (United States)

    Phelps, Nicholas B D; Patnayak, Devi P; Jiang, Yin; Goyal, Sagar M

    2012-12-01

    Viral hemorrhagic septicemia virus (VHSV) is a highly contagious and pathogenic virus of fish. The virus infects more than 70 fish species worldwide, in both fresh and salt water. A new viral strain (VHSV-IVb) has proven both virulent and persistent, spreading throughout the Great Lakes of North America and to inland water bodies in the region. To better understand the geographic distribution of the virus, we used a modified real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for high-throughput testing of fish for VHSV. The assay was shown to be twice as sensitive as the gold standard, virus isolation, and did not cross react with other viruses found in fish. In addition, the diagnostic turnaround time was reduced from 28 to 30 d for virus isolation to 2-4 d for rRT-PCR. To demonstrate the usefulness of the rRT-PCR assay, 115 high-priority water bodies in Minnesota were tested by both methods from April 2010 to June 2011. All survey sites tested negative for VHSV by both methods. The survey results have informed fisheries managers on the absence of VHSV in Minnesota and have better prepared them for the eventual arrival of the disease. In addition, the results demonstrate the value of this rRT-PCR as a surveillance tool to rapidly identify an outbreak so that it can be controlled in a timely manner.

  7. Detection of norovirus in oyster by real time RT-PCR%应用荧光定量RT-PCR法检测牡蛎中诺如病毒

    Institute of Scientific and Technical Information of China (English)

    邓丽丽; 刘巍; 莫建光; 谭冬梅; 谢镇国; 唐振柱; 董柏青

    2011-01-01

    目的 建立牡蛎样品中的诺如病毒检测方法.方法 优化甘氨酸缓冲液处理牡蛎匀浆液,摸索PEG沉淀浓缩病毒的浓度,提取病毒RNA,采用荧光定量RT-PCR方法进行诺如病毒检测.应用优化后的方法检测105份牡蛎样本,并用核苷酸序列测定法对阳性PCR产物进行验证.结果 甘氨酸缓冲液pH值为4.5,PEG沉淀浓度为16%时的诺如病毒富集提取效果最好,该方法的诺如病毒检出限为1.17×102个质粒拷贝.应用该方法对未知样本进行检测阳性率达14.29%,所得阳性PCR产物经核苷酸序列测序证实为诺如病毒.结论 本研究方法可以应用于牡蛎中的诺如病毒检测,南宁市海鲜市场上的部分牡蛎含有诺如病毒.%Aim To develop an assay for detection of norovirus in oysters. Methods The viral RNA was extracted and purified by using an optimized protocol of virus elution with glysine buffer and virus precipitation with polyethylene glycol (PEG). The RNA extracts were analyzed for norovirus using realtime RT-PCR. Positive PCR products were sequenced to further confirm the presence of virus. Results When the pH of glysine buffer was 4.5 and the concentration of PEG was 16%,higher levels of norovirus could be detected,with the detection limit of 1.17×102 copies. By this protocol,15 (14.29%) out of the 105 samples were tested positive. All the positive PCR products were confirmed as norovirus by sequence analysis. Conclusion The method adopted in this study is applicable for use in screening norovirus in oysters. The result shows that norovirus is present in oysters from aquatic markets in Nanning.8

  8. Design and analysis of Q-RT-PCR assays for haematological malignancies using mixed effects models

    DEFF Research Database (Denmark)

    Bøgsted, Martin; Mandrup, Charlotte; Petersen, Anders;

    research use and needs qualit control for accuracy and precision. Especially the identification of experimental variations and statistical analysis has recently created discussions. The standard analytical technique is to use the Delta-Delta-Ct method. Although this method accounts for sample specific...... variations such as RNA purification, it does not account for other experimental effects as variations in cDNA synthesis, amplification efficiency and assay variations. To obtain an assessment of the accuracy and precision of the assays a novel approach for the statistical analysis of Q-RT-PCR has been...... developed based on a linear mixed effects model for factorial designs. The model consists of an analysis of variance where the variation of each fixed effect of interest and identified experimental and biological nuisance variations are split. Hereby it accounts for varying efficiency, inhomogeneous...

  9. No control genes required: Bayesian analysis of qRT-PCR data.

    Directory of Open Access Journals (Sweden)

    Mikhail V Matz

    Full Text Available BACKGROUND: Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process. RESULTS: In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts. Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the "classic" analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests. CONCLUSIONS: Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been

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

    Science.gov (United States)

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

    2017-07-01

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

  11. The development of real time RT-PCR for avian influenza virus H10N8%H10N8禽流感病毒Real time RT-PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    薄洪; 高荣保; 李晓丹; 张烨; 邹淑梅; 朱云; 刘琳玉; 王大燕; 舒跃龙

    2015-01-01

    目的 建立针对H10N8禽流感病毒特异、灵敏的real time RT-PCR检测方法.方法 通过比对现有H10N8病毒血凝素(HA)和神经氨酸酶(NA)全长基因序列,分别针对HA和NA基因设计引物和探针6套(P1-P6)和3套(P7-P9),建立鉴别H10N8禽流感病毒real time RT-PCR方法,分别对筛选的引物进行特异性、灵敏性和临床标本检测适应性等评价.结果 特异性检测显示6套针对HA的引物和探针中的3套能够检测H10N8病毒,其中2套(P5和P6)与其他非H10亚型流感病毒无交叉反应;3套针对NA的引物和探针均能检测H10N8病毒,其中2套(P7和P9)与其他非N8亚型流感病毒无交叉反应.灵敏性检测显示,P5和P6以及P7和P9均能检出最大稀释度为109的H10N8病毒RNA,重复性显示4组的Ct值CV均<3%.使用P6和P9对3例H10N8病例呼吸道标本份进行real time RT-PCR检测均为阳性,其中2例病毒分离阳性,4份病毒分离阳性的活禽相关环境样本检测均为阳性.结论 建立了H10N8禽流感病毒荧光定量PCR检测方法,该方法具有良好的特异性和敏感性.%Objective Establish real time RT-PCR methods for detecting H10N8 subtype of avian influenza virusesand evaluate the sensitivity and specificity of this methods.Methods Compared the existing hemagglutinin (HA) and neuraminidase (NA) gene sequences of H10N8 subtype respectively,primers and probes of HA and NA were designed.Six sets of HA primers and probes (P1-P6) and three sets of NA primers and probes (P7-P9) were evaluated for specificity,sensitivity and adaptability.Results All primers and probes were used to detect the H10N8 subtype and other subtypes influenza viruses.Two sets of primers and probes (P5 and P6) showed no cross reaction with other H10 subtype influenza viruses.Three sets of NA primers and probes (P7,P8 and P9) could detect the H10N8 virus.P7and P9 showed no reaction with non-N8 subtype influenza viruses.Sensitivity assay showed that P5,P6,P7 and P9 could detect

  12. 应用细菌磁颗粒实时荧光RT-PCR检测南瓜花叶病毒%Application of bacterial magnetic particles real-time RT-PCR for detecting Squash mosaic virus

    Institute of Scientific and Technical Information of China (English)

    孙宁; 邓丛良; 么磊; 陈继峰

    2010-01-01

    @@ 南瓜花叶病毒(Squash mosaic virus,SqMV)隶属豇豆花叶病毒属(Comovirus),是一种严重危害黄瓜、西葫芦等葫芦科作物的病毒,在自然界可通过种子、机械摩擦和昆虫介体传播.目前对于该病毒的检测主要采用鉴别寄主反应、血清学检测、电镜观察、分子生物学检测等.实时荧光RT-PCR(real-time fluorescent RT-PCR)作为一种灵敏度更高的检测技术,近年来被广泛应用于植物病毒的 检测[1].

  13. 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

    Directory of Open Access Journals (Sweden)

    María G Barbás

    2012-03-01

    Full Text Available 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.Durante la pandemia de influenza A (H1N1, la OMS recomendó algoritmos y protocolos de detección del virus mediante RT-PCR en tiempo real. El objetivo del presente estudio fue evaluar el desempeño del equipo que comercializa la empresa Roche, Real Time Ready Influenza A/H1N1 Detection Set (junio de 2009, en comparación con el protocolo de RT-PCR en tiempo real de los CDC. La sensibilidad global del ensayo de Roche para la detección del gen Inf A en presencia o ausencia del gen H1 fue 74,5 %. La sensibilidad para la detección de muestras positivas solo para el gen Inf A (ausencia del gen H1 fue 53,3 % y la sensibilidad para la detección de muestras positivas para H1N1 (presencia del gen Inf A y cualquier otro gen porcino fue 76,4 %. La especificidad fue 97,1 %. Existe una nueva versión del equipo (noviembre 2009 que, según se ha descrito, presenta buena sensibilidad en comparación con otros ensayos moleculares para detectar H1N1 pandémica.

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

    OpenAIRE

    Yue-Jiao Ma; Xiao-Hong Sun; Xiao-Yan Xu; Yong Zhao; Ying-Jie Pan; Cheng-An Hwang; 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 ...

  15. Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa).

    Science.gov (United States)

    Song, Hao; Dang, Xin; He, Yuan-Qiu; Zhang, Tao; Wang, Hai-Yan

    2017-01-01

    The veined rapa whelk Rapana venosa is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in R. venosa. For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in R. venosa for use as internal controls for qRT-PCR. In this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1α (EF-1α), α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1α subcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms. Of the 13 candidate genes tested, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. For the normalization of a single specific tissue, we suggested that EF-1α and NDUFA7 are the best combination in gonad, as well as COX1 and RL28 for intestine, EF-1α and RL5 for kidney, EF-1α and COX1 for gill, EF-1α and RL28 for Leiblein and mantle, EF-1α, RL5, and NDUFA7 for liver, GAPDH, PPIA, and RL28 for hemocyte. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured

  16. 实时荧光定量RT-PCR检测鼻咽癌Survivin mRNA基因表达%Detection of Survivin mRNA in nasopharyngeal carcinoma by real-time fluorescence quantitative RT-PCR

    Institute of Scientific and Technical Information of China (English)

    Shengmiao Fu; Junhong Cai; Zhihua Tu; Yutian Wang; Liqun Deng; Zhu Liang; Zhenqun Lin; Xuanju Gong

    2008-01-01

    Objective:To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeal carcinoma (NPC) tissues.Methods:The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA,a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established,in which chronic nasopharyngitis patients' nasopharynx tissues treated as control group.Results:The expression of Survivin mRNA all could be detected either in CNE-2 cells,NPC tissues or in chronic nasopharyngitis patients' nasopharynx tissues,and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients' nasopharynx tissues,the difference was significant (P<0.01).The expression of Survivin mRNA could be detected both in stage Ⅰ+Ⅱ and stage Ⅲ+Ⅳ NPC,and there was no significant difference in relative quantifications of gene expression between these two groups (P>0.05).There was no relationship between Survivin mRNA expression and age and sex of NPC patients (P>0.05).Conclusion:Real time fluorescence quantitative RT-PCR is a rapid,effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues.The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.

  17. Real-time quantitative RT-PCR assay for detection of Maedi-visna virus%梅迪-维斯纳病毒实时荧光定量PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    张太翔; 肖越强; 杨慧; 凌宗帅; 孙涛; 袁涛; 徐彪; 梁成珠; 刘文鹏; 孙军; 王洪兵

    2011-01-01

    为建立检测梅迪-维斯纳病毒(MVV)的TagMan实时荧光定量PCR方法,本研究根据MVV核苷酸保守序列设计引物和探针.以梯度稀释的含有MVV目的扩增片段的重组质粒作为标准品,进行定量PCR反应.结果显示:5.0×105~5.0×101,拷贝范围内定量PCR均有"S"型扩增曲线,检测灵敏度为50拷贝.对羊的其他病毒核酸均无扩增反应.本研究建立的实时定量PCR方法,灵敏度高,特异性好,在NM的快速检测中具有良好的应用前景.%A real-time quantitative RT-PCR assay was developed for detection of Maedi-visna virus (MVV). Primers and probes were designed based on the sequence of MW by Primer Express 2.0. The recombinant plasmid containing the target sequence was constructed to detect the sensitivity and prepare the standard curve. The real-time RT-PCR assay had a detection limit of 50 copies, with a dynamic range of detection between 5 × 105 copies to 5 × 101 copies. The primers and probe were specific for MVV and did not react with other virus, including Capripox virus, pseudorabies virus and Foot-mouth disease virus.The real-time RT-PCR assay described here with high sensitivity, specificity and accuracy is considered to be a powerful tool for the rapid detection and quantification of MVV.

  18. 马尔堡病毒的实时荧光RT-PCR检测方法研究%Study on real time RT - PCR detection method for Marburg virus

    Institute of Scientific and Technical Information of China (English)

    洪烨; 邓燕凤; 郑夔; 相大鹏; 黄吉城; 戴俊; 师永霞; 李小波; 幸芦琴; 郭波旋

    2011-01-01

    Objective To set up real time RT - PCR detection method for Marburg virus. Methods Some representative nucleic acid segment of Marburg virus as positive control were synth esized, and several primers, probes and reaction system of real time RT - PCR were designed to explore the best detection condition. The PCR condition was optimized to improve the sensitivity and specificity of the assay. Results The specificity of the assay for real time RT - PCR for Marburg virus was high and there was no cross reactions with Dengue virus, Japanese encephalitis virus and Chikungunya virus. The sensitivity of the assay was 100 gene copies per test. Conclusion This method is suitable for laboratory detection of Marburg virus because of its high sensitivity and specificity.%目的 建立马尔堡病毒的实时荧光RT-PCR检测方法.方法 人工合成马尔堡病毒特异性核酸序列作为阳性对照模板,设计实时荧光RT-PCR引物、探针并构建反应体系,对反应条件进行优化,验证该方法的特异性、灵敏度.结果 建立的马尔堡病毒实时荧光RT-PCR检测方法对马尔堡病毒核酸检测有高度特异性,与1型~4型登革病毒、日本脑炎病毒和基孔肯雅病毒均无交叉反应,检测灵敏度为102拷贝/反应.结论 该方法灵敏度高、特异性强,适用于对马尔堡病毒的快速检验.

  19. Selection of reference genes for normalisation of real-time RT-PCR in brain-stem death injury in Ovis aries

    Directory of Open Access Journals (Sweden)

    Fraser John F

    2009-07-01

    Full Text Available Abstract Background Heart and lung transplantation is frequently the only therapeutic option for patients with end stage cardio respiratory disease. Organ donation post brain stem death (BSD is a pre-requisite, yet BSD itself causes such severe damage that many organs offered for donation are unusable, with lung being the organ most affected by BSD. In Australia and New Zealand, less than 50% of lungs offered for donation post BSD are suitable for transplantation, as compared with over 90% of kidneys, resulting in patients dying for lack of suitable lungs. Our group has developed a novel 24 h sheep BSD model to mimic the physiological milieu of the typical human organ donor. Characterisation of the gene expression changes associated with BSD is critical and will assist in determining the aetiology of lung damage post BSD. Real-time PCR is a highly sensitive method involving multiple steps from extraction to processing RNA so the choice of housekeeping genes is important in obtaining reliable results. Little information however, is available on the expression stability of reference genes in the sheep pulmonary artery and lung. We aimed to establish a set of stably expressed reference genes for use as a standard for analysis of gene expression changes in BSD. Results We evaluated the expression stability of 6 candidate normalisation genes (ACTB, GAPDH, HGPRT, PGK1, PPIA and RPLP0 using real time quantitative PCR. There was a wide range of Ct-values within each tissue for pulmonary artery (15–24 and lung (16–25 but the expression pattern for each gene was similar across the two tissues. After geNorm analysis, ACTB and PPIA were shown to be the most stably expressed in the pulmonary artery and ACTB and PGK1 in the lung tissue of BSD sheep. Conclusion Accurate normalisation is critical in obtaining reliable and reproducible results in gene expression studies. This study demonstrates tissue associated variability in the selection of these

  20. Validation of a norovirus multiplex real-time RT-PCR assay for the detection of norovirus GI and GII from faeces samples.

    LENUS (Irish Health Repository)

    Jones, S

    2011-01-01

    Norovirus is a leading cause of infectious non-bacterial gastroenteritis. The virus is highly contagious and has multiple modes of transmission, presenting a growing challenge to hospital-based healthcare. In this study, a total of 120 stool samples are tested for the presence of norovirus GI and GII by the Roche two-step Lightcycler 2.0 assay incorporating primers and probes produced by TIB Molbiol, and the results are compared with results from the National Virus Reference Laboratory. The Roche\\/TIB Molbiol assay produced 51 positive results and 69 negative results. Discrepancy analysis was performed for six conflicting results using a second real-time polymerase chain reaction (PCR) assay (Roche\\/TIB Molbiol) and this confirmed that four of the five discrepant positive results were true positives. A single discrepant negative result generated by the Roche assay remained negative using the second assay. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated to be 98%, 98.6%, 98.0% and 98.6%, respectively. Melting curve analysis was used to differentiate genogroups I and II and this showed that 92% of strains belonged to genogroup II.

  1. Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR).

    Science.gov (United States)

    Leusch, F D L; Van den Heuvel, M R; Laurie, A D; Chapman, H F; Gooneratne, S Ravi; Tremblay, L A

    2005-01-01

    A method to quantify induction of vitellogenin (Vtg) mRNA in adult male mosquitofish was developed. Male mosquitofish were exposed to 0, 1, 20 and 250 ng l(-1) 17beta-oestradiol (E(2)) for 4 and 8 days in static exposures, and liver Vtg mRNA and 18S rRNA expression were quantified in duplex RT-PCR. Liver 18S rRNA expression was very consistent among individuals, and there was a highly significant increase in Vtg mRNA expression after exposure of mosquitofish for just 4 days at 250 ng l(-1) E(2). Lower doses did not induce Vtg mRNA expression even at 4 or 8 days. This method could be used as a rapid test to detect exposure of mosquitofish to oestrogenic chemicals. Further work is needed to determine if increased Vtg mRNA levels in male mosquitofish induce Vtg synthesis, and to determine the usefulness of the method in field sampling.

  2. 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

    Directory of Open Access Journals (Sweden)

    Rashi Gautam

    2016-01-01

    Full Text Available 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 q

  3. 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.

    Science.gov (United States)

    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

  4. Virological course of hepatitis A virus as determined by real time RT-PCR: Correlation with biochemical, immunological and genotypic profiles

    Institute of Scientific and Technical Information of China (English)

    Zahid Hussain; Apiradee Theamboonlers; Yong Poovorawan; Premashis Kar; Bhudev C Das; Syed A Husain; Sunil K Polipalli; Tanzeel Ahmed; Nargis Begum; Subhash Medhi; Alice Verghese; Mohammad Raish

    2006-01-01

    AIM: To undertake analysis of hepatitis A viral load,alanine aminotransferase (ALT), and viral genotypes with duration of viremia, and to correlate these parameters with CD4+/ CD8+ lymphocyte populations that control cell-mediated immunity.METHODS: Cell counts were carried out using fresh whole blood collected in EDTA vials using a fluorescence activated cell sorter. Hepatitis A virus (HAV) RNA was extracted from blood serum, reverse transcribed into cDNA and quantified by Real-Time polymerase chain reaction and was genotyped.RESULTS: Among 11 patients, 10 could be analyzed completely. Of these, 3 had severe acute hepatitis (s-AH) and the remainder had a self-limited acute hepatitis A (AHA), with one patient with fulminant disease (encephalopathy Grade Ⅳ) dying on the 4th d. The ALT level was significantly higher both in AHA (1070.9 ± 894.3; P = 0.0014) and s-AH (1713.9 ± 886.3; P = 0.001) compared to normal controls (23.6 ± 7.2). The prothrombin time in s-AM patients (21.0 ± 2.0; P = 0.02) was significantly higher than in AHA (14.3 ± 1.1;P = 0.44). The CD4+/CD8+ ratio in AHA patients (1.17 ± 0.11; P = 0.22) and s-AH (0.83 ± 0.12; P = 0.0002) were lower than seen in normal healthy controls (1.52).Self-limited cases had peak viral load at the beginning of analysis while in s-AH patients this occurred at the 15th or 30th d. In acute and severe groups, one patient each belonged to genotype ⅠA, with the remaining 8 cases belonging to genotype ⅢA. The only fulminant hepatic failure case belonged to genotype ⅠA. HAV viral load and ALT values collected during the entire course of the selflimited infection were directly correlated but this was not the case for s-AH patients.CONCLUSION: Based on a small-scale study, the persistently higher viral load of s-AH might be due to diminished cellular immunity and hemolysis. The duration of viremia was dependent on the host, as the viral genotype had no apparent role in clinical outcome of AVH and s-AH cases.

  5. RT-PCR and Northern blot analysis in search for a putative Paramecium beta-adrenergic receptor.

    Science.gov (United States)

    Płatek, A; Wiejak, J; Wyroba, E

    1999-01-01

    RT-PCR and Northern blot analysis were performed in order to search for a putative beta-adrenergic receptor (beta-AR) in Paramecium using several beta2-adrenergic-specific molecular probes. Under strictly defined RT-PCR conditions DNA species of expected molecular size about 360 bp were generated with the primers corresponding to the universal mammalian beta2-AR sequence tagged sites (located within the 4th and the 6th transmembrane regions of the receptor). This RT-PCR product hybridized in Southern blot analysis with the oligonucleotide probe designed to the highly conservative beta2-AR region involved in G-proteins interaction and located within the amplified region. Northern hybridization was performed on Paramecium total RNA and mRNA with human beta2-AR cDNA and two oligonucleotide probes: the first included Phe 290 involved in agonist binding (Strader et al., 1995) and the second was the backward RT-PCR primer. All these probes revealed the presence of about 2 kb mRNA which is consistent with the size of beta2-AR transcripts found in higher eukaryotes.

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

    Science.gov (United States)

    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.

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

    OpenAIRE

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

    2011-01-01

    Background : Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conven...

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

    Directory of Open Access Journals (Sweden)

    Yue-Jiao Ma

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  10. Real-Time RT-PCR检测手足口病肠道病毒方法的建立及应用%Establishment and Application of Real-time RT-PCR Method for Detection of Enteroviruses of Hand, Foot and Mouth Disease

    Institute of Scientific and Technical Information of China (English)

    罗建平; 曹正安; 贺放晴; 石文松

    2010-01-01

    目的 建立手足口病肠道病毒的Real-Time RT-PCR法,为手足口病病毒快速、准确检测提供-种新方法.方法 采用卫生部(2009年版,以下简称)推荐的RT-PCR法对湖南省株洲市2009年3-8月间发生的82例临床诊断手足口病患儿的82份标本进行人肠道病毒、柯萨奇病毒A16型和肠道病毒71型特异性核酸的检测,同时采用实时荧光定量RT-PCR(Real-Time Quantitative RT-PCR,简称Real-Time RT-PCR)进行复检,比较二者的实验结果,分析Real-TimeRT-PCR的准确度和灵敏度.结果 Real-Time RT-PCR检测82份标本肠道病毒、柯萨奇病毒A16型、肠道病毒71型,与RT-PCR比较,二者差异有统计学意义(λ=4.09,P<0.05),Real-Time RT-PCR法检出率明显高于RT-PCR法.结论 本研究建立了一种灵敏、特异、简单易行的Real-Time RT-PCR检测手足口病肠道病毒的方法,为手足口病病毒的快速诊断提供了新的方法.

  11. Development and evaluation of a SYBR Green real-time RT-PCR assay for evaluation of cytokine gene expression in horse.

    Science.gov (United States)

    Sánchez-Matamoros, A; Kukielka, D; De las Heras, A I; Sánchez-Vizcaíno, J M

    2013-01-01

    Cytokine secretion is one of the main mechanisms by which the immune system is regulated in response to pathogens. Therefore, the measurement of cytokine expression is fundamental to characterizing the immune response to infections. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) is widely used to measure cytokine mRNA levels, but assay conditions should be properly evaluated before analyzing important equine infections through relative quantification of gene expression. The aim of this study was to develop and evaluate a set of RT-qPCR assays for a panel of the most common cytokines in horses involved in innate and adaptive immune responses. Eight cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-10, IL-12, TNFα, IFNβ and IFNγ) and a housekeeping gene (β-actin) were detected and amplified with the same annealing temperature in a SYBR Green RT-qPCR assay of samples of mitogen-stimulated peripheral blood mononuclear cells from a healthy horse and whole blood from a horse infected with African horse sickness virus. The method gave good efficiency for all genes tested, allowing quantification of relative expression levels. These SYBR Green RT-qPCR assays may be useful for examining cytokine gene expression in horses in response to exposure to economically important pathogens.

  12. Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in Iris. lactea var. chinensis Roots under Cadmium, Lead, and Salt Stress Conditions

    Directory of Open Access Journals (Sweden)

    Chun-Sun Gu

    2014-01-01

    Full Text Available Quantitative real time PCR (RT-qPCR has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC, tubulin alpha-5 (TUBLIN, eukaryotic translation initiation factor (EIF-5A, translation elongation factor EF1A (EF1α, translation elongation factor EF1B (EF1b, actin11 (ACTIN, and histone H3 (HIS, in Iris. lactea var. chinensis (I. lactea var. chinensis root when the plants were subjected to cadmium (Cd, lead (Pb, and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (Ct values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed that EIF-5A and UBC were the most stable reference genes across all of the tested samples, while TUBLIN was unsuitable as internal controls. I. lactea var. chinensis is tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

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

    Science.gov (United States)

    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.

  14. Reference gene selection for quantitative real-time RT-PCR normalization in Iris. lactea var. chinensis roots under cadmium, lead, and salt stress conditions.

    Science.gov (United States)

    Gu, Chun-Sun; Liu, Liang-qin; Xu, Chen; Zhao, Yan-hai; Zhu, Xu-dong; Huang, Su-Zhen

    2014-01-01

    Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1 α ), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), in Iris. lactea var. chinensis (I. lactea var. chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (C t ) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed that EIF-5A and UBC were the most stable reference genes across all of the tested samples, while TUBLIN was unsuitable as internal controls. I. lactea var. chinensis is tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

  15. Reverse-transcription PCR (RT-PCR).

    Science.gov (United States)

    Bachman, Julia

    2013-01-01

    RT-PCR is commonly used to test for genetic diseases and to characterize gene expression in various tissue types, cell types, and over developmental time courses. This serves as a form of expression profiling, but typically as a candidate approach. RT-PCR is also commonly used to clone cDNAs for further use with other molecular biology techniques (e.g., see Oligo(dT)-primed RT-PCR isolation of polyadenylated RNA degradation intermediates and Circularized RT-PCR (cRT-PCR): analysis of RNA 5' ends, 3' ends, and poly(A) tails).

  16. The study on quantitative expression of CD44v6mRNA by real-time RT-PCR with the micro-metastases of gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Daorong Wang; Xunliang Liu; Guoyu Chen; Yi Miao; Jianguo Xia

    2005-01-01

    Objective: To study the expression of CD44 correlation and the ability of metastasis of tumor cells in gastric carcinoma, and to find the correlation of the quantitative of CD44V6mRNA and the histology expression of CD44v6 in tumors with the clinic-pathologic features, and to make the quantitative expression of CD44v6mRNA. Methods: Twenty patients with gastric carcinoma, 4patients with gastritis, and 10 apparently healthy controls were recruited. Blood samples were obtained before surgery. 10 days after surgery, the blood samples were obtained again. Serum CD44v6mRNA in all cases was measured by real-time quantitative PCR. Results:Serum CD44V6mRNA was detectable in 20 of 20( 100% ) gastric carcinoma cases, The expression level ranged from 4.9 × 102 copies/μg RNA to 3.2 × 10s copies/μg RNA, the average levels of peripheral blood was 3.9 × 104 copies/μg RNA, The expression level of peripheral blood of gastric cancer after curative operation ranged from 5.5 × 100 copies/μg RNA to 7.6 × 103 copies/μg RNA. After curative operation the expression level was decreased markedly. Conclusion: Serum CD44v6mRNA is expressed in the peripheral blood of gastric carcinoma patients. The expression level of CD44V6mRNA is obviously decreased after curative operation. An elevated level of CD44v6mRNA may serve as an indicator of lymph node metastasis (especially early metastasis) and bad prognosis in patients with gastric carcinoma.

  17. Bluetongue virus detection by real-time RT-PCR in Culicoides captured during the 2006 epizootic in Belgium and development of an internal control.

    Science.gov (United States)

    Vanbinst, T; Vandenbussche, F; Vandemeulebroucke, E; De Leeuw, I; Deblauwe, I; De Deken, G; Madder, M; Haubruge, E; Losson, B; De Clercq, K

    2009-06-01

    After the emergence of bluetongue (BT) in Belgium in 2006, two types of entomological surveys were initiated, the one to identify the local vector species, and the other to study their population dynamics. In the vector study, Culicoides were captured near farms with recently infected cattle or sheep; in the population study Culicoides were captured in two meadows situated in the BT-affected region. A total of 130 pools of parous, non-blood engorged female midges (with a mean of 7.5 midges per pool) were analysed with real-time reverse transcription PCR (RT-qPCR) targeting bluetongue virus (BTV) segment 5. To ensure the RNA integrity of the samples, all pools were also tested in a second RT-qPCR targeting Culicoides 18S rRNA, which served as an internal control. Seventeen pools with negative results for both 18S and BTV were excluded, most of which originated from the population survey. In the vector survey near outbreak sites, female midges of the obsoletus complex, including C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, dominated the black-light trap collections with 19 of 89 pools being BTV-positive. Moreover, all the collections from the vector survey included at least one positive pool of the obsoletus complex compared with only 20% collections (C. obsoletus/C. scoticus) in the population survey. The current study also revealed the presence of BTV RNA in one of five pools of C. pulicaris females captured near recent BT outbreaks, suggesting that this species might have played a role in transmission. Finally, the use of RT-qPCR for the recognition of new potential BTV vector species and the impact of an appropriate monitoring method and internal control are discussed.

  18. Reference gene selection for quantitative real-time RT-PCR normalization in the half-smooth tongue sole (Cynoglossus semilaevis at different developmental stages, in various tissue types and on exposure to chemicals.

    Directory of Open Access Journals (Sweden)

    Conghui Liu

    Full Text Available Quantitative real time RT-PCR has been described as the most sensitive method for the detection of low abundance mRNA. To date, no reference genes have been screened in the half-smooth tongue sole (Cynoglossus semilaevis. The aim of this study was to select the most stable genes for quantitative real-time RT-PCR. Eight housekeeping genes (18S, TUBA, B2M, ACTB, EF1A, GAPDH, RPL17 and UBCE were tested at different developmental stages, in different tissues, and following exposure to the drug SB-431542. Using geNorm, BestKeeper and NormFinder software, GAPDH/B2M, GAPDH/18S and UBCE/GAPDH were identified as the most suitable genes from samples taken of different developmental stages while 18S/RPL17 were consistently ranked as the best reference genes for different tissue types. Furthermore, TUBA/B2M, TUBA/UBCE and B2M/TUBA were found to be the most suitable genes in samples treated with the drug, SB-431542 by geNorm, BestKeeper and NormFinder respectively. Across both different developmental stages and tissue types, the combination of 18S and GAPDH was the most stable reference gene analyzed by Ref-Finder. To test and verify the screened reference genes, the expression profiles of LEFTY-normalized to the combination of GAPDH/18S and ACTB were presented. These results will be useful for future gene-expression studies in the half-smooth tongue sole.

  19. A one-step, triplex, real-time RT-PCR assay for the simultaneous detection of enterovirus 71, coxsackie A16 and pan-enterovirus in a single tube.

    Directory of Open Access Journals (Sweden)

    Shiyin Zhang

    Full Text Available The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD, which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71 and coxsackie A16 (CA16 are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001-0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.

  20. Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A

    Directory of Open Access Journals (Sweden)

    Van Zeveren Alex

    2006-10-01

    Full Text Available Abstract Background An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor γ coactivator 1α (PPARGC1A is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types. Results The mRNA expression stability of 10 reference genes was determined. The expression of RPL13A and SDHA appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes (ACTB, TBP and TOP2B, the results not only showed that the mRNA expression of PPARGC1A was significantly higher in each of the longissimus dorsi muscle samples than in backfat (P Conclusion This study provides a new set of reference genes (ACTB, TBP and TOP2B suitable for normalization of real-time RT-PCR data of backfat and longissimus dorsi muscle in the pig. The obtained PPARGC1A expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass.

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

    Directory of Open Access Journals (Sweden)

    Thonur Leenadevi

    2012-03-01

    Full Text Available Abstract 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.

  2. Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR

    Directory of Open Access Journals (Sweden)

    Kundu Jiban K

    2010-07-01

    Full Text Available Abstract Background Reference genes are commonly used as the endogenous normalisation measure for the relative quantification of target genes. The appropriate application of quantitative real-time PCR (RT-qPCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. For this purpose, seven reference genes were investigated in tissues of the most important cereals (wheat, barley and oats. Titre of Barley yellow dwarf virus (BYDV was determined in oats using relative quantification with different reference genes and absolute quantification, and the results were compared. Results The expression of seven potential reference genes was evaluated in tissues of 180 healthy, physiologically stressed and virus-infected cereal plants. These genes were tested by RT-qPCR and ranked according to the stability of their expression using three different methods (two-way ANOVA, GeNorm and NormFinder tools. In most cases, the expression of all genes did not depend on abiotic stress conditions or virus infections. All the genes showed significant differences in expression among plant species. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, beta-tubulin (TUBB and 18S ribosomal RNA (18S rRNA always ranked as the three most stable genes. On the other hand, elongation factor-1 alpha (EF1A, eukaryotic initiation factor 4a (EIF4A, and 28S ribosomal RNA (28S rRNA for barley and oat samples; and alpha-tubulin (TUBA for wheat samples were consistently ranked as the less reliable controls. The BYDV titre was determined in two oat varieties by RT-qPCR using three different quantification approaches. There were no significant differences between the absolute and relative quantifications, or between quantification using GAPDH + TUBB + TUBA +18S rRNA and EF1A + EIF4A + 28S rRNA. However, there were discrepancies between the results of individual assays

  3. Real-time Quantitative RT-PCR Assay for Specific Identification of Panthera tigris Materials%基于TaqMan探针的虎制品实时荧光定量PCR的特异性鉴定

    Institute of Scientific and Technical Information of China (English)

    张太翔; 凌宗帅; 赵立娜; 袁涛; 徐彪; 梁成珠; 刘文鹏; 孙军; 张艺兵

    2012-01-01

    根据虎线粒体细胞色素b核苷酸保守序列设计引物和探针,建立了一种快速鉴定虎制品的TaqMan实时荧光定量PCR方法.以梯度稀释的含有目的扩增片段的重组质粒作为标准品,进行定量PCR反应.结果显示,5.0×105~5.0×101拷贝/μL范围内定量PCR均有“S”型扩增曲线,检测灵敏度为50拷贝/μL.对豹、狮子、猫等7种非虎哺乳动物DNA样本的检测结果均为阴性.本研究建立的实时荧光定量PCR方法具有灵敏度高、特异性和重复性好、方便经济的特性,在虎制品的检测与鉴定中具有良好的应用前景.%Primers and probes were designed based on the conservative sequence of cytochondriome cytochrome b, a realtime quan quantitative RT-PCR assay was developed for indentification of Panthera tigris. The recombinant plasmid containing the target sequence was constructed to detect the sensitivity and prepare the standard curve. The real-time RT-PCR assay had a detection limit of 50 copies/μL, and with a dynamic range of detection between 5 × 105 to 5 × 101 copies /μL. The primers and probe were specific for Panthera tigris' DNA and did not react with other non- Panthera tigris ' DNA. This real-time RT-PCR assay with high sensitivity, specificity and accuracy is considered to be a powerful tool for the rapid indentification and quantification of Panthera tigris.

  4. 玉米褪绿斑驳病毒实时荧光RT-PCR检测方法研究%Detection of Maize chlorotic mottle virus by real-time fluorescent RT-PCR

    Institute of Scientific and Technical Information of China (English)

    闻伟刚; 张建成; 崔俊霞; 张颖

    2011-01-01

    Maize chlorotic mottle virus ( MCMV) is a quarantine pests issued by Chinese government. In this study, accord to the conservative sequences of the coat protein genes of different MCMV isolates, a real - time fluorescent RT - PCR method was established based on specific primers and TaqMan probe. Specific studies have shown that for the two MCMV strains from different sources, a typical amplification curve can be obtained, and not for the Wheat streak mosaic virus, Maize rough dwarf virus and Maize dwarf mosaic virus. Sensitivity comparison found that the detection limit can reach 10 ~5 dilution, which is 100 times higher than the regular RT - PCR method. Thus, the real - time fluorescent RT - PCR is a rapid, sensitive and highly specific method for the detection of MCMV.%玉米褪绿斑驳病毒(Maize chlorotic mottle virus,MCMV)是我国对外公布的检疫性有害生物.本研究根据该病毒外壳蛋白基因的保守序列,设计得到特异性引物及Taqman荧光探针,建立了MCMV的实时荧光RT-PCR方法,并对其灵敏度与特异性进行了研究.该方法针对2个不同来源的毒株均能得到典型扩增曲线,而没有从小麦线条花叶病毒、玉米粗缩病毒和玉米矮花叶病毒的RNA得到扩增曲线,表明引物与荧光探针具有良好的特异性.针对玉米褪绿斑驳病毒RNA不同稀释度样品,实时荧光RT-PCR检测低限达到10-5稀释度,检测灵敏度要比普通RT-PCR高出100倍.因此,本研究建立的MCMV实时荧光方法具有特异性强、灵敏度高和快速有效的优点.

  5. Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for RT-PCR and microarray analysis.

    Science.gov (United States)

    Kashofer, Karl; Viertler, Christian; Pichler, Martin; Zatloukal, Kurt

    2013-01-01

    Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application

  6. Detection of Citrus tatter leaf virus (CTLV) by real-time RT-PCR%运用实时荧光RT-PCR技术检测柑橘碎叶病毒

    Institute of Scientific and Technical Information of China (English)

    刘科宏; 周常勇; 宋震; 周彦; 李中安; 唐科志

    2009-01-01

    柑橘碎叶病是由橘碎叶病毒(Citrus tatter leaf virus,CTLV)引起的一种重要的柑橘病害,为更快、更准确的检测CTLV,合成了一对特异性引物ASG-Pf和ASG-Pr,建立了运用SYBR Green Ⅰ荧光染料法检测CTLV的实时荧光RT-PCR体系,并对该体系的特异性、灵敏性和适用性进行了测试.结果表明.该检测体系能特异的检出CTLV,对测试的衰退病毒、温州蜜柑萎缩病毒和鳞皮病毒都不能检出;灵敏度比常规PCR高100倍;适用性广,可检测出多种柑橘类植物中的CTLV.实时荧光RT-PCR检测整个过程完全闭管,无需PCR后处理,且SYBR Green Ⅰ荧光染料成本较低.适用于检测柑橘体内含量较低的CTLV病毒.%Citrus tatter leaf disease caused by citrus tatter leaf virus (CTLV) is an economically important systemic disease of citrus in the world. So it is essential to develop a rapid and reliable method to detect CTLV. Using a pair of primers ASG-Pf and ASG-Pr, a real-time RT-PCR system based on SYBR Green Ⅰ dye has been established to detect CTLV. The specificity, sensitivity and applicability of this system were detected. The results showed that only CTLV could be detected, while Citrus tristeza virus (CTV), Satsuma dwarf virus (SDV)and Citrus psorosis virus (CPV) can not be detected. The sensitivity of real-time PCR is higher than that of conventional PCR by 100 fold, and it can detect CTLV isolates from different kinds of citrus cuhivars. Real-time RT-PCR doesn't need post-PCR handling of the amplified products such as electrophoresis, staining and imaging by completing the whole process within a single tube, and SYBR Green Ⅰ dye needs low cost, so it is suitable to detect CTLV.

  7. Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae).

    Science.gov (United States)

    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

    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. 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. 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.

  8. Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae.

    Directory of Open Access Journals (Sweden)

    Rumei Li

    Full Text Available 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.

  9. Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses.

    Science.gov (United States)

    Hadjinicolaou, Andreas V; Farcas, Gabriella A; Demetriou, Victoria L; Mazzulli, Tony; Poutanen, Susan M; Willey, Barbara M; Low, Donald E; Butany, Jagdish; Asa, Sylvia L; Kain, Kevin C; Kostrikis, Leondios G

    2011-04-01

    Emerging infectious diseases have caused a global effort for development of fast and accurate detection techniques. The rapidly mutating nature of viruses presents a major difficulty, highlighting the need for specific detection of genetically diverse strains. One such infectious agent is SARS-associated coronavirus (SARS-CoV), which emerged in 2003. This study aimed to develop a real-time RT-PCR detection assay specific for SARS-CoV, taking into account its intrinsic polymorphic nature due to genetic drift and recombination and the possibility of continuous and multiple introductions of genetically non-identical strains into the human population, by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV S, E, M and N genes. These were applied in simple, reproducible duplex and multiplex real-time PCR assays on 25 post-mortem samples and constructed RNA controls, and they demonstrated high target detection ability and specificity. This assay can readily be adapted for detection of other emerging and rapidly mutating pathogens.

  10. Establishment of RT-PCR and SYBR Green Real-time PCR for detection of Flavivirus in mosquito%检测蚊虫感染黄病毒属病毒Real-time PCR方法的建立

    Institute of Scientific and Technical Information of China (English)

    周晓俊; 朱淮民; 邱璐

    2008-01-01

    目的 建立SYBR Green Real-time PCR检测和筛选黄病毒属病毒方法. 方法 参照文献报道的通用引物,以JEV cDNA 和DEN cDNA为模板,建立RT-PCR和SYBR Green Real-time PCR,检测和筛选黄病毒属病毒,并比较两者的敏感性. 结果 此黄病毒属引物适合两种反应体系,SYBR Green Real-time PCR方法的敏感性是RT-PCR方法的100倍,最低检出病毒浓度为0.5×10-2 PFU/ml. 结论 建立的两种方法均可用于黄病毒属病毒检测,以SYBR Green Real-time PCR方法具有更高的敏感性,对于黄病毒属病毒初筛检测具有应用价值.

  11. 扎伊尔型埃博拉病毒的实时荧光RT-PCR检测方法研究%Development of Real-time RT-PCR assay for detection of Zaire Ebolavirus

    Institute of Scientific and Technical Information of China (English)

    李阿茜; 李伟红; 李建东; 张硕; 曲靖; 李川; 张全福; 梁米芳; 李德新

    2014-01-01

    目的 建立扎伊尔型埃博拉病毒的核酸检测方法,以期用于埃博拉出血热临床标本的检测.方法 针对扎伊尔型埃博拉病毒核蛋白和糖蛋白基因设计引物和探针,建立单重和双重实时荧光RT-PCR检测方法,利用体外转录病毒RNA和埃博拉病毒系列参考品RNA评价其敏感性,利用马尔堡病毒、健康人、登革热患者和发热伴血小板减少综合征患者血清评价其特异性.结果 所建立的实时荧光RT-PCR检测方法扩增效率在95%~105%,可特异性地检测扎伊尔型埃博拉病毒核蛋白和糖蛋白基因,与马尔堡病毒、登革热和发热伴血小板减少综合征病毒均无交叉反应,体外转录的病毒RNA可检出10~100拷贝/μl.双重检测方法通过细胞培养的扎伊尔型埃博拉病毒RNA验证,可检出100 pfu/ml病毒.结论 本研究建立的检测扎伊尔型埃博拉病毒的实时荧光RT-PCR方法具有良好的特异性和敏感性,可用于埃博拉出血热临床标本的检测.%Objective To establish a method for Zaire Ebolavirus (ZEBOV) RNA detection and laboratory diagnosis of suspected ZEBOV infected cases.Methods Primers/probe sets for Nucleoprotein (NP) and Glycoprotein(GP) detection were designed and used to develop monoplex and duplex real-time RT-PCR assays.The sensitivity was evaluated by in vitro transcribed RNA and ZEBOV RNA reference,and the specificity was identified by Marburg virus,healthy human sera and sera from Dengue fever patients and Severe fever with thrombocytopenia syndrome (SFTS) patients.Results The developed real-time RT-PCR assay scould be used for ZEBOV NP and GP detection specifically,and the amplification efficiency was 95% -105%.There was no cross reaction with Marburg virus,Dengue fever virus and SFTS virus.The sensitivity was evaluated with serial dilutions of synthesized viral RNAs,and the results showed that in vitro transcribed RNA of 10-100 copies/μl could be detected.The developed duplex

  12. SYBR Green Ⅰ荧光定量RT-PCR检测埃博拉病毒方法的建立%Development of SYBR Green Ⅰ real-time RT-PCR for the Detection of Ebola Virus

    Institute of Scientific and Technical Information of China (English)

    刘阳; 王水明; 刘学辉; 马志永; 史子学; 马玉堃; 王皓婷; 王宗尧; 邵东华; 魏建超; 王少辉; 李蓓蓓

    2012-01-01

    In order to establish a rapid and accurate method for the detection of Ebola virus (EBOV), the primers used in SYBR Green I real-time RT-PCR were designed based on the EBOV NP gene sequences published in GenBank. The SYBR Green I real-time RT-PCR was established and optimized for the detection of EBOV. The EBOV RNA that was transcribed in vitro was used as a template. The sensitivity of this method was found to reach 1, 0 × 102 copies/μL and the detection range was 102~1010. No cross reaction with RNA samples from Marburg virus, Dengue virus, Xinjiang hemorrhagic fever virus, Japanese encephalitis virus, Influenza virus (H1N1 and H3N2) and Porcine reproductive and respiratory syndrome virus E genomic RNA was found.. The method would be useful for the detection and monitoring of EBOV in China.%为了建立一种快速准确的检测埃博拉病毒(EBOV)亚型的方法.本研究根据GenBank中公布的EBOV NP基因序列,通过设计引物和优化反应条件,建立了一种SYBR Green Ⅰ荧光定量RT-PCR检测方法检测EBOV.以体外转录的EBOV RNA为模板进行试验,该方法检测的灵敏度可以达到1.0×102个拷贝/μL,检测范围达到9个数量级为102~1010,可检测5种亚型EBOV.建立的方法对马尔堡病毒(MARV)、登革病毒(DENV)、新疆出血热病毒(XHFV)、乙型脑炎病毒(JEV)、流感病毒(H1N1和H3N2)和猪繁殖和呼吸综合征病毒(PRRSV)E基因组RNA无非特异性扩增.本文将荧光定量RT-PCR技术用于埃博拉病毒的定量检测中,并且建立了EBOV SYBRGreen Ⅰ荧光定量RT-PCR检测方法.

  13. The RT-PCR Analysis of Lignocellulytic Biodegradation-related Gene Expression of Phanerochaete chrysosporium

    Institute of Scientific and Technical Information of China (English)

    Jiang Mingfeng(江明锋); Zhang Yizheng

    2004-01-01

    Expression of lignocellulytic biodegradation-related genes of Phanerochaete chrysosporium grown in 10 kinds of defined media for 5 days and on fir wood chip for 2 to 8 weeks is analyzed by using the RT-PCR method. The result shows that an individual gene of lip gene family responds differently to different nutrient factors. The expression of lipD gene can be promoted by molecular O2 but suppressed by Mn2+. The influence of nitorgen is not the controlling factor for lipD gene expression. No clear relationship is found between nutrient factors and the expression of lipA gene which may be regulated by several nutrient factors through a complex system. Mnp3 gene is not strongly regulated by Mn2+ and other nutrient factors. It can be expressed in different media. CBHI gene family can not be expressed in the presence of glucose as the sole carbon source. Glx expression is regulated by Mn2+ and molecular O2, and depressed when Mn2+ concerntration goes up to 300 mg/L. The transcription patterns of lip gene family grown on fir wood chip are shown to be markedly different from those patterns in defined media. The expression of single lip gene changes with colonized time. No difference is observed between the expression pattern of mnp, cbh, glx gene in defined media and fir wood chips.

  14. Proteomic analysis and qRT-PCR verification of temperature response to Arthrospira (Spirulina platensis.

    Directory of Open Access Journals (Sweden)

    Wang Huili

    Full Text Available 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

  15. Proteomic analysis and qRT-PCR verification of temperature response to Arthrospira (Spirulina) platensis.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  17. Assessment of GM-CSF receptors by real-time RT-PCR on cell lines expressing high and low affinity receptors and their relation to cytotoxic effect of chimeric protein (StxA1-GM-CSF

    Directory of Open Access Journals (Sweden)

    Habibi Roudkenar M.

    2007-06-01

    Full Text Available Immunotoxins, which are composed of both the cell targeting and the cell killing moieties are the new approach for targeted therapy of human disease .In all immunotoxins that GM-CSF has been used as cell targeting; only cell lines expressing high affinity receptor have been used for cytotoxicity studies. In the present study, various cell lines expressing high and low affinity receptors were used for assessment of the cytotoxic effect of hybrid chimeric protein. The expression of GM-CSF receptor (GM-CSFR was quantified by real-time RT- PCR. The cell lines K562 and THP1 expressing high affinity receptor and MC-7, PC-3 and DU145 expressing low affinity receptor were used for this study. The chimeric hybrid protein was found to be toxic for various cell lines used in this investigation and cytotoxicity was more effective in cell lines bearing high affinity receptors. Overall, our results showed that the recombinant hybrid protein could have wide range of application on various cancer cell lines even cells bearing low affinity receptors for GM-CSF.

  18. Expression patterns of WT-1 and Bcr-Abl measured by TaqMan quantitative real-time RT-PCR during follow-up of leukemia patients with the Ph chromosome

    Institute of Scientific and Technical Information of China (English)

    CHEN Zi-xing陈子兴; Jaspal Kaeda; Sue Saunders; John M Goldman

    2004-01-01

    Background This study was designed to quantitatively measure WT-1 expression levels in patients with chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) during follow-up and to clarify the value of WT-1 as a molecular marker in minimal residual disease monitoring.Methods The TaqMan quantitative real-time RT-PCR method was established by using cloned WT-1 cDNA or synthesized oligonucleotides resembling WT-1 cDNA fragments in limit dilution as template until a stable and reliable standard curve was obtained. In a 25-month follow-up, the transcriptional levels of WT-1, Bcr-Abl, and Abl gene, were quantitatively measured in bone marrow cells from 25 CML or acute lymphoblastic leukemia (ALL) patients with the Ph chromosome. In addition, the expression of these genes in 40 samples of normal peripheral blood was also examined using the same method. The ratios of WT-1/Abl and Bcr-Abl/Abl were both plotted, and the two expression patterns were compared as well as their clinical significance.Results The levels of WT-1 expression in normal peripheral blood were detectable. In CML and Ph positive ALL patients, WT-1 expression levels changed in parallel with the Bcr-Abl expression pattern as the disease progressed or responded to effective treatment.Conclusion WT-1 expression provides a novel molecular marker in addition to Bcr-Abl for monitoring minimal residual disease (MRD) and targeting therapy in Ph chromosome-positive leukemia patients.

  19. Real time analysis under EDS

    Energy Technology Data Exchange (ETDEWEB)

    Schneberk, D.

    1985-07-01

    This paper describes the analysis component of the Enrichment Diagnostic System (EDS) developed for the Atomic Vapor Laser Isotope Separation Program (AVLIS) at Lawrence Livermore National Laboratory (LLNL). Four different types of analysis are performed on data acquired through EDS: (1) absorption spectroscopy on laser-generated spectral lines, (2) mass spectrometer analysis, (3) general purpose waveform analysis, and (4) separation performance calculations. The information produced from this data includes: measures of particle density and velocity, partial pressures of residual gases, and overall measures of isotope enrichment. The analysis component supports a variety of real-time modeling tasks, a means for broadcasting data to other nodes, and a great degree of flexibility for tailoring computations to the exact needs of the process. A particular data base structure and program flow is common to all types of analysis. Key elements of the analysis component are: (1) a fast access data base which can configure all types of analysis, (2) a selected set of analysis routines, (3) a general purpose data manipulation and graphics package for the results of real time analysis. Each of these components are described with an emphasis upon how each contributes to overall system capability. 3 figs.

  20. Establishment of a SYBR Green Ⅰ-based real-time RT-PCR for rapid detection of duck Tembusu virus%鸭坦布苏病毒SYBR GreenⅠ实时荧光定量检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    万春和; 朱海侠; 施少华; 黄瑜; 程龙飞; 傅光华; 陈红梅

    2013-01-01

    A pair of specific primers targeted to non structure gene 5 (NS5) of duck Tembusu virus was designed and a SYBR Green Ⅰ fluorescent based real-time RT-PCR (RRT-PCR) was developed for the quantization of duck Tembusu virus.The detection limit of RRT-PCR was 2.74 × 102plasmid copies.The melting curve analysis using SYBR Green Ⅰ dye showed one specific peak,a melting temperature (Tm) was (86.23±0.18) ℃,and no primer-dimers peak was observed.No amplification was detected from unrelated virus samples by this method,such as avian influenza virus,duck hepatitis virus type 1,avian paramyxovirus type 1,egg drop syndrome virus,duck reovirus.Fine reproducibility was obtained for detecting plasmid DNA with intra-assay of 0.52%1.48% and inter-assay of 0.71%-2.21%.The real-time PCR method developed in this study will be useful for rapid laboratory diagnosis and epidemiology investigation for duck Tembusu virus.%根据鸭坦布苏病毒(Duck Tembusu virus,DFV) NS5基因序列特征设计引物,建立基于SYBR Green Ⅰ检测模式的实时荧光定量RT-PCR(real-time RT-PCR,RRT-PCR),该方法检测DFV NS5基因2.74×103~2.74×10 7拷贝/μL反应范围内有很好的线性关系.扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,Tm值为(86.23±0.18)℃,对禽流感病毒、鸭肝炎病毒、鸭源禽1型副黏病毒、鸭减蛋综合征病毒、番鸭呼肠孤病毒核酸均无阳性信号扩增,可重复性好,组内变异系数为0.52%~1.48%,组间变异系数0.71%~2.21%.检测速度快,从样本处理到报告结果仅需4h.

  1. 鸭黄病毒荧光定量RT-PCR检测方法的建立及应用%Development and application of real-time RT-PCR assay for duck flavivirus

    Institute of Scientific and Technical Information of China (English)

    高凤; 于可响; 马秀丽; 李玉峰; 王友令; 李建亮; 崔言顺

    2013-01-01

    A real-time RT-PCR method was developed for the detection of duck flavivirus (DFV). The specific primers and TaqMan probe were designed and synthesized according to the E gene sequence of duck flavivirus. The standard curve (Y=-3. 39 X + 43. 18,r = 0. 973) was plotted based on the linear relationship between the amount of plasmid DNA and cycle threshold (Ct). This method was specific for duck flavivirus(DFV) but not for duck hepatitis virus,avian influenza virus,Newcastle disease virus,adenovirus,duck plague virus and porcine Japanese encephalitis virus. The detection limit reached 1. 9 TCID50 of the virus under the optimized conditions. Tissues in five ducks after 86 h artificial infected with DFV were tested positive by this method, without parts of cecal tonsils. And the positive detection rate of the method was consistent with routine virus isolation method. These results indicated that the real-time RT-PCR approach provides a powerful diagnostic tool with high sensitivity and specificity for the identification and quantitation of DFV and the whole process of the test could be completed within 3 hours.%根据鸭黄病毒E蛋白基因序列设计了1对特异性引物和TaqMan探针,建立了实时荧光定量RT-PCR检测鸭黄病毒的方法.根据舍鸭黄病毒目的扩增片段的质粒拷贝数与定量反应Ct值的关系,绘制了标准曲线(y=-3.39 X+43.18,r=0.973).该方法具有特异性,对鸭病毒性肝炎病毒、禽流感病毒、新城疫病毒、腺病毒、鸭瘟病毒、猪乙脑病毒的核酸都没有扩增反应.敏感性试验显示,建立的此方法最低可检出1.9个TCID50病毒核酸.该方法对5只健康雏鸭人工感染86 h后的组织器官样品(盲肠扁桃体除外)的检测结果均为阳性,与病毒分离鉴定的阳性符合率为100%.结果表明,该方法真实可靠,而且从核酸提取到报告检测结果耗时不超过3h,为鸭黄病毒病提供了一种敏感特异的定量检测方法.

  2. Development and Application of Real-time RT-PCR Assay for Chicken Infectious Bronchitis Virus%鸡传染性支气管炎病毒Real-time RT-PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    王鹏; 高峰; 王园; 杨莹; 路红; 周双海; 刘凤华

    2013-01-01

    The objective of this study was to develop a Real-time RT-PCR assay of chicken Infectious Bronchitis Virus (IBV) and therefore to detect the load IBV quantitatively. A fragment of N gene in IBV was amplified with RT-PCR method, and was cloned into the vector pEASY-T3. Then, the recombinant plasmid containing the N gene fragment was constructed. The standard curve and corresponding linear regression equation of IBV nucleic acid level were developed by Real-time PCR based on SYBR Green I with the recombinant plasmid. This method showed a high specificity and had a detection limit of 5.58X102 copies/p,L, and its coefficient of variations was less than 3.2% in the reproducible assays. The virus nucleic acid in tissue samples from chickens inoculated experimentally with IBV M41 strain was quantitatively determined with the established Real-time RT-PCR. The detection results showed that the load of IBV in the kidney was more than that in bronchus and lung after inoculation, and the virus load in the bronchus and lung on 3 days post-inoculation (DPI) were higher than those on 7 DPI and 10 DPI. Moreover, the correlation between the clinical manifestations and viral load was confirmed. The results indicated that this Real-time RT-PCR assay was of high specificity, sensitivity and reproducibility, and could be used for the quantitative detection of IBV.%为定量检测鸡传染性支气管炎病毒(IBV)载量,建立IBV的Real-time RT-PCR方法.用RT-PCR方法扩增出IBV的N基因片段,并克隆到pEASY-T3载体中,构建成含有N基因片段的重组质粒.应用该重组质粒进行SYBR Green I Real-time PCR,建立了定量检测IBV核酸的标准曲线与直线回归方程,该方法显示:特异性强,检测下限至少达到5.58× 102拷贝/μL,其重复性试验的变异系数小于3.2%;用建立的方法对实验接种IBV M41株的雏鸡组织中的病毒核酸进行了定量检测,检测结果显示:攻毒后肾脏中IBV含量高于支气管和肺脏,支气管

  3. Real-time analysis keratometer

    Science.gov (United States)

    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.

  4. Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

    Directory of Open Access Journals (Sweden)

    Marone Maria

    2001-01-01

    Full Text Available We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative analysis are described here, together with an example from a study on the effects of TGF-&bgr;1 in TF-1 cells.

  5. Real-time RT-PCR quantification of pregnancy-associated plasma protein-A mRNA abundance in bovine granulosa and theca cells: effects of hormones in vitro.

    Science.gov (United States)

    Aad, Pauline Y; Voge, Justin L; Santiago, Consuelo A; Malayer, Jerry R; Spicer, Leon J

    2006-11-01

    Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins -2, -4 and -5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1-5 mm) and large (LG) (8-22 mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24 h. Cells were treated with various concentrations (3-500 ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24 h (Experiments 1-10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected (P>0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect (P>0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased (P<0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles.

  6. 戊型肝炎病毒TaqMan Real-time RT-PCR法的建立及应用%Establishment and application of TaqMan real-time RT-PCR for the detection of hepatitis E virus

    Institute of Scientific and Technical Information of China (English)

    孟庆玲; 邱丰; 沈立萍; 毕胜利

    2012-01-01

    目的 建立灵敏、特异、稳定的戊型肝炎病毒(HEV) TaqMan Real-time PCR检测方法.方法 根据GenBank中的HEV相关序列,选取HEV基因组ORF2的保守区域设计合成特异性引物和探针,建立TaqMan HEV Real-time RT-PCR检测体系,评价体系的特异性、敏感度和稳定性,并应用于临床样本的检测.结果 本研究建立的HEV Real-time RT-PCR检测体系最低检测极限达到10个拷贝/反应,重复性实验Ct值的变异系数(CV)最大为1.53%,并且该体系能特异检测出戊肝临床样本中的HEV,其拷贝数从1.87×104拷贝/ml到8.12×106拷贝/ml不等.结论 成功建立特异性强、灵敏度高的HEV Real-time RT-PCR检测方法,应用于临床样本检测时取得了良好效果,为HEV分子病原学诊断打下基础.%Objective To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis E virus (HEV).Methods According to the references,primers-probe sets which were located in ORF2,the conservative part of HEV genome were designed and therefore we established a HEV TaqMan real-time RT-PCR assay with great performance of specificity,sensitivity and reproducibility.And then it was used in the detection of HEV RNA in clinical samples.Results The HEV Real-time RT-PCR assay established in this study were able to detect HEV RNA with a detection limit of 10 copies/reaction.When the detection of a same sample was repeated for several times,coefficients of variation (CV) was all less than 1.53%.Our data also suggested that there were 1.87 × 106-8.12 × 109 RNA copies in 1ml of the clinical samples.Conclusion The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HEV RNA.It was applied successfully in the pathogen detection of clinical samples.

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

    Science.gov (United States)

    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.

  8. Detection and Differentiation of Vaccine and Wild-type Strains of PPRV by Duplex Real-time RT-PCR%实时荧光RT-PCR鉴别小反刍兽疫病毒疫苗株和野毒株

    Institute of Scientific and Technical Information of China (English)

    李林; 吴晓东; 刘春菊; 王清华; 包静月; 王志亮

    2016-01-01

    To differentiate vaccine virus and wild-type peste des petits ruminants virus(PPRV)by duplex real-time RT-PCR,one pair of primers and two TaqMan probes were designed based on the comparison of PPRV genome sequences available in GenBank. The duplex real-time RT-PCR results showed that the detection limits for vaccine strain and wild-type strains were both 10 copies per reaction. The developed assay also showed high ampliifcation efifciency,linearity and speciifcity in discriminating the two PPRV types. A total of 136 clinical samples tested by the conventional RT-PCR were run again by the duplex real-time RT-PCR. The results were conifrmed subsequently by sequencing the positive conventional RT-PCR products. This reliable and rapid duplex real-time RT-PCR assay would be feasible and attractive for diagnostic laboratories,both for clinical purpose in active surveillance and for epidemiological investigations.%为快速鉴别小反刍兽疫病毒的疫苗株和野毒株,本研究通过分析GenBank中已发布的小反刍兽疫病毒全基因组序列,设计了1对通用引物以及疫苗株和野毒株特异性探针各1条,建立了小反刍兽疫病毒鉴别实时荧光RT-PCR方法。结果显示,该方法对疫苗株和野毒株的检测灵敏度均可以检测至10拷贝/反应,在108至101拷贝/反应之间具有良好的线性关系,扩增效率均接近1。该方法具有良好的特异性,能够区分不同谱系的野毒株株和疫苗株。使用该方法对136份临床疑似样品检测,检出的阳性数量与普通RT-PCR相同,并且测序后的区分结果也相同。本方法的建立为该病的实验室鉴别检测和流行病学调查提供了技术支持。

  9. Real-time flutter analysis

    Science.gov (United States)

    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.

  10. Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer.

    Science.gov (United States)

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tiwari, Ashish; Skillman, Ashley; Nam, Bora; Chambers, Thomas M; Tsai, Yun-Long; Ma, Li-Juan; Yang, Pai-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2014-10-01

    Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated

  11. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

    Science.gov (United States)

    Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

    2012-01-01

    Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

  12. Development and application of a real-time TaqMan RT-PCR assay for detection of duck hepatitis virus type 1%I型鸭肝炎病毒 TaqMan 荧光定量 RT-PCR 方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    刘海燕; 赵丽丽; 牛银杰; 祝明皓; 刘胜旺; 陈洪岩

    2015-01-01

    Objective To develop a real-time RT-PCR assay ( rRT-PCR) for efficient detection of duck hepatitis virus type 1 ( DHV-I) .Method According to the different gene sequences of DHV-I from different provinces download from NCBI and to find the conserved sequences.One pair of the specific primers and one TaqMan probe were designed. Then reaction parameters were optimized to develop a real-time RT-PCR assay ( rRT-PCR) .Results This developed rRT-PCR assay could detect 20 template copies of RNA, and its sensitivity was higher than that of the conventional RT-PCR. This rRT-PCR assay was found to be specific and able to detect DHV-I, and no positive results were observed when nucleic acid from Muscovy duck parvovirus, goose parvovims, Newcastle disease and avian influenza virus, egg drop syndrome virus, reticuloendotheliosis virus, duck Tembusu virus, poultry intestinal arc virus were used as rRT-PCR templates.The results of this developed rRT-PCR assay used for 100 duck clinical samples showed a positive rate of 92%, indicating that DHV exists in duck group of Jiangsu province in China.Conclusion This rRT-PCR assay can be used as a rapid tool for detection of DHV-I.%目的:建立快速诊断I型鸭肝炎病毒的荧光定量RT-PCR方法。方法根据NCBI下载的20个来自我国不同省份的的I型鸭肝炎病毒的基因序列,找出其保守序列,设计合成一对引物和一条TaqMan探针,进行条件优化,检测其特异性,敏感性,稳定性。结果该方法敏感性达20拷贝,比常规PCR敏感性高。其特异性强,对番鸭细小病毒(MDPV),鹅细小病毒(GPV),新城疫病毒(NDV)和禽流感(AIV),鸭减蛋综合征病毒(EDSV),禽网状内皮组织增生症病毒(REV),鸭坦布苏病毒(DTMUV),禽呼肠弧病毒(ARV)8种病毒的检测均为阴性,I型鸭肝炎病毒检测结果为阳性。用建立的方法检测了江苏徐州采集100份样品,阳性率为92%。说明I型鸭

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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.

  15. Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A and Asia-1 foot-and-mouth disease virus lineages circulating in the Middle East

    DEFF Research Database (Denmark)

    Reid, Scott M.; Mioulet, Valerie; Knowles, Nick J.

    2014-01-01

    transcription polymerase chain reaction (rRT-PCR) assays using primer/probe sets designed from the VP1 coding region of the virus genomes were developed for the specific detection of serotype O, A and Asia-1 FMD viruses (FMDVs) circulating in the Middle East. These assays were evaluated using representative...... three primer/probe sets detected the RNA from homotypic viruses and no cross-reactivity was observed with heterotypic viruses. Similar results were obtained using both single- and multiplex assay formats. Using plasmid standards, the minimum detection level of these tests was found to be lower than two...

  16. Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus

    DEFF Research Database (Denmark)

    Li, Yiping; Bang, Dang Duong; Handberg, Kurt

    2005-01-01

    -time RT-PCR is needed to a suitable internal control. We thus investigated the expression pattern of six chicken genes, including P-actin, 28S rRNA, 18S rRNA, glyceral dehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP) and beta-2-microglobulin, in chicken embryo (CE) cell cultures...... and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2...... virus-specific RNA load in CE cell cultures. Thus, beta-actin was suggested as a suitable internal control in studying gene expression as well as virus-specific RNA load in CE cell after IBDV infection....

  17. Quantification of human telomerase reverse transcriptase mRNA in gastric cancer with real-time fluorescent quantitative RT-PCR%胃癌中人端粒酶逆转录酶mRNA的实时定量检测

    Institute of Scientific and Technical Information of China (English)

    胡丽华; 陈凤花; 李一荣; 王琳

    2004-01-01

    目的建立一种实时荧光定量逆转录-聚合酶链反应(RT-PCR)方法,检测胃癌组织中hTERT mRNA 的表达,探讨hTERT的表达水平与胃癌的关系及其在胃癌诊断中的价值.方法采用Taqman技术与LightCycler荧光定量PCR仪对35例胃癌及其相应切缘组织中hTERT mRNA的表达进行实时定量检测.将hTERT与GAPDH拷贝数之比的100倍作为标准化hTERT(NhTERT).结果 (1)胃癌及其相应切缘组织中NhTERT分别为6.27±0.89、0.93±0.18,两者之间差异有统计学意义(t=12.76,P<0.01).(2)胃癌组织中hTERT mRNA的表达水平与组织的分化程度密切相关(P<0.01),而与患者的年龄、性别、肿瘤的大小、定位以及pTNM分期无相关性.结论实时荧光定量RT-PCR 方法能对hTERT mRNA进行准确、高效的定量.hTERT mRNA的实时定量检测可能有助于胃癌的早期诊断.

  18. Comparison of protocols for the analysis of type 1 porcine reproductive and respiratory syndrome virus by RT-PCR using oral fluids.

    Science.gov (United States)

    Gibert, Elisa; Martín-Valls, Gerard; Mateu, Enric

    2017-05-01

    The detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OF) by quantitative real-time polymerase chain reaction (qRT-PCR) is gaining increasing popularity. However, the different steps leading to a result have not been extensively evaluated. The aim of the present study was to examine the effect on the performance of qRT-PCR with different sampling materials, conditions of storage of the OF, the need for centrifuging OF, as well as to compare RNA extraction methods and PCR mixes. For the assays, pen-based oral fluids were used, which were pooled and spiked in a serial dilution (up to genotype 10(0) TCID50/mL) of type 1 PRRSV isolate 3267. Centrifugation at 15,000g for 15min resulted in an increase in sensitivity (1-2 PCR cycles) that was significant (PPCR Kit PCR mix reagents were more sensitive for the detection of PRRSV using a purified plasmid as standard, but LSI VetMAX PRRSV EU/NA PRRSV reagents resulted in a slightly better sensitivity with OF (pPCR. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. A competitive RT-PCR method for the quantitative analysis of cytokine mRNAs in mouse tissues.

    Science.gov (United States)

    Zhou, N M; Matthys, P; Polacek, C; Fiten, P; Sato, A; Billiau, A; Froyen, G

    1997-03-01

    The authors describe the design and validation of a competitive RT-PCR method for the efficient and reproducible quantitation of mRNA molecules of IFN-gamma, TNF-alpha, IL-4 and IL-10 in mouse spleen RNA extracts. Before being subjected to RT-PCR, the RNA extracts were supplemented with internal control RNAs (IC-RNAs), which were constructed by inserting DNA fragments in the cDNA of the respective cytokines. The efficiency of amplification of the target and the IC-RNA was shown to remain equal over a wide range of cycle numbers. Reproducibility was such that differences in mRNA contents that were greater than 17% could be detected between two RNA samples run in parallel. Normal mouse spleen tissue was found to contain 10(7)-10(8) molecules of TNF-alpha, IFN-gamma, IL-4 and IL-10 mRNA per micrograms total RNA extracted. Injection of animals with anti-CD3 antibody, a well-known cytokine inducer, resulted in a moderate increase in TNF-alpha and IL-10 mRNA levels (14- and 24-fold, respectively), and in a substantially greater increase in the levels of mRNA for IL-4 and IFN-gamma (199- and 851-fold, respectively). These results demonstrate an accurate and reliable quantitation of cytokine mRNA levels in animal tissues.

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

    Directory of Open Access Journals (Sweden)

    Alok Arun

    Full Text Available 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

  1. Establishment and in-house validation of stem-loop RT PCR method for MicroRNA398 expression analysis

    Directory of Open Access Journals (Sweden)

    Timotijević Gordana S.

    2015-01-01

    Full Text Available MicroRNAs (miRNAs belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by “stem-loop” primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for “stem loop” primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as “stem loop” RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs. [Projekat Ministarstva nauke Republike Srbije, br. 173005 i br. ON-173028

  2. Analysis of real-time vibration data

    Science.gov (United States)

    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.

  3. Analysis of Porcine Reproductive and Respiratory Syndrome Virus Isolates from Eastern China with RT-PCR-RFLP%猪繁殖与呼吸综合征病毒华东地区分离株RT-PCR-RFLP分析

    Institute of Scientific and Technical Information of China (English)

    邓雨修; 姜平; 李玉峰; 王先炜; 蒋文明; 汤景元

    2004-01-01

    Porcine reproductive and respiratory syndrome virus(PRRSV) isolates identified in samples from 50 field cases originated from eastern China herds were studied. ORF5 gene of PRRSV isolates was amplified by reverse transcript polymerase chain reaction(RT-PCR) and restriction fragment length polymorphism (RFLP) patterns for enzymes Mlu Ⅰ, Hinc Ⅱ and Sac Ⅱ were determined on these ORF5 genes. The RFLP pattern obtained from 39 isolates was 2-2-1(78%), 3 isolates was 1-1-1(6%) and 8 isolates identified in 2003 was 1-3-1 (16%). The results showed PRRSV strains have variations and existed no less than 3 gene subtypes in the area.

  4. 禽流感病毒通用型及H5亚型套式荧光RT-PCR检测方法研究%Development of Nested Real-time RT-PCR for Detection of Influenza A Viruse and Subtype H5

    Institute of Scientific and Technical Information of China (English)

    杨素; 廖秀云; 廖明; 沙才华; 徐海聂; 曹伟胜; 陈伟生

    2012-01-01

    Combining nested PCR (NP-PCR) with real-time RT-PCR,superimposing advantages of sensitivity and specificity of these two methods,establwhed nested real-time RT-PCR for detecting AIV and subtype H5, which was more sensitive than conventional real-time RT-PCR. Basing on the conserved AIV NP and HA gene downloaded from GenBank. Designed nested real-time RT-PCR primers and TaqMan probes for detecting AIV and subtype H5. Target segments recombined plasmids were constructed,and positive controls were prepared with technology of in vitro transcription. Nested real-time RT-PCR was established after reaction mixtures and condition screened out. Test results indicated that this established method can specific detect AIV and H5 subtype, 19 of 30 fish aquaculture water samples were presented positive results, multitudinous samples can be detected simultaneously within 5 h. It is a good method for detecting trace AIV from samples of the natural environment, with distinguishing feature of high sensitivity,good specificity,high throughout and rapidity.%将套式PCR与荧光RT PCR技术相结合,叠加这两种技术在敏感性和特异性方面的优势,建立敏感性高于常规荧光RT-PCR的套式荧光RT-PCR检测方法.从GenBank下载禽流感病毒NP基因和HA基因序列,通过比对选取较为保守的片段,设计AIV通用型及H5亚型套式荧光RT-PCR引物和TaqMan探针;利用体外转录技术制备阳性对照,构建目的片段重组质粒;经反应体系和反应条件的优化,建立套式荧光RT-PCR检测方法.试验结果表明,该方法可特异性地检测禽流感病毒及H5亚型AIV,在30份鱼类养殖水样中检测到19份阳性样品,具有较高的敏感性和良好的特异性;具有高通量、快速的特点,可对大批量样品同时进行检测,试验耗时仅需5h,是自然环境样品中极微量禽流感病毒检测的好方法.

  5. Analysis of Mitochondrial RNA-Processing Defects in Patient-Derived Tissues by qRT-PCR and RNAseq.

    Science.gov (United States)

    Kopajtich, Robert; Mayr, Johannes A; Prokisch, Holger

    2017-01-01

    Transcription of the mitochondrial genome yields three large polycistronic transcripts that undergo multiple endonucleolytic processing steps, before resulting in functional mRNAs, tRNAs, and rRNAs. Cleavage of the large precursor transcripts is mainly performed by the RNase P complex and RNase Z that cleave mitochondrial pre-tRNAs at their 5' and 3' ends respectively. Most likely there are additional enzymes involved that still await identification and characterization. Defects in mitochondrial RNA processing have been associated with human disease. There are published cases of patients carrying mutations in either HSD17B10/MRPP2 (encoding a subunit of RNase P complex) or ELAC2 (coding for RNase Z). In addition, several mtDNA mutations within tRNA genes have been shown to affect RNA processing. Here, we describe detailed protocols for analyzing RNA processing of mitochondrial tRNAs, in particular their 3'-ends that are processed by RNase Z. These protocols should serve as a guide to extract RNA for quantitative real-time PCR and RNAseq analysis.

  6. Determination of the expressions of inflammatory cytokines in mouse vaginal mucosa with real-time relative quantitative RT PCR%实时相对定量反转录PCR检测小鼠阴道黏膜炎症因子的表达

    Institute of Scientific and Technical Information of China (English)

    李亮助; 杨瑜; 袁松华; 贲银银; 徐建青; 张晓燕

    2011-01-01

    目的 建立实时相对定量反转录聚合酶链反应(real-time RQ RT-PCR)方法,能快速准确地检测各种炎症因子在阴道黏膜的转录表达情况,从而评价包括杀微生物剂在内的生殖道或直肠黏膜用候选药物的刺激毒性.方法 以Eppendorf Mastercycler ep realplex实时荧光定量PCR仪为检测平台,选择小鼠β-actin基因为内参,建立基于SYBR Green Ⅰ荧光染料的real-time PCR检测方法,对白介素2(Interleukin 2,IL-2)、IL-4、IL-6、IL-10、IL-17A、肿瘤坏死因子α(Tumor necrosis factor α,TNF-α)、γ干扰素(Interferon γ,IFN-γ)等炎症因子在生殖道黏膜的mRNA转录水平,同时进行检测,并用2--⊿⊿Ct方法计算实验组小鼠目的基因相对于空白组小鼠的表达差异情况.用微量样本多指标流式蛋白定量技术(Cytometric Bead Array,CBA)对结果进行验证.结果 使用real-time RQ RT-PCR能对小鼠阴道黏膜炎症因子的表达情况进行快速检测,结果与CBA检测结果相吻合.结论 该方法快速、灵敏、特异性强、价格低廉、高通量.可用于杀微生物剂等生殖道或直肠黏膜用候选药物的临床前安全性评价工作.%Objective To determine expressions of inflammatory cytokines in mouse vaginal mucosa which are important endpoints in the preclinical safety evaluation of mucosa applied agents including microbicide. Methods Relative gene expressions of seven cytokines in vaginal mucosa. Including interleukin (ID-2. IL-4. IL-6, IL-10. IL-17A, tumor necrosis factor (TNF)- α and interferon (IFN)- γ,were analyzed using real-time relative quantification reverse transtription PCR (real-time RQ RT PCR) and the 2-△△Ct method. The results were verified by Cytometric Bead Array (CBA). Results The expressions of inflammatory cytokines in mouse vaginal mucosa can be quantified by real-time RQ RT PCR in a rapid and accurate way, the result of which is corroborate with that of CBA. Conclusions The developed real-time RQ RT

  7. Real-time analysis of telemetry data

    Science.gov (United States)

    Kao, Simon A.; Laffey, Thomas J.; Schmidt, James L.; Read, Jackson Y.; Dunham, Larry L.

    1987-01-01

    This paper descibes a knowledge-based system for performing real-time monitoring and analysis of telemetry data from the NASA Hubble Space Telescope (HST). In order to handle asynchronous inputs and perform in real time the system consists of three or more separate processes, which run concurrently and communicate via a message passing scheme. The data management process gathers, compresses, and scales the incoming telemetry data befoe sending it to the other tasks. The inferencing process uses the incoming data to perform a real-time analysis of the state and health of the Space Telescope. The I/O process receives telemetry monitors from the data management process, updates its graphical displays in real time, and acts as the interface to the console operator. The three processes may run on the same or different computers. This system is currently under development and is being used to monitor testcases produced by the Bass Telemetry System in the Hardware/Software Integration Facility at Lockheed Missile and Space Co. in Sunnyvale, California.

  8. 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.

    Science.gov (United States)

    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.

  9. Quantitative RT-PCR analysis of differentially expressed genes in Quercus suber in response to Phytophthora cinnamomi infection.

    Science.gov (United States)

    Ebadzad, Ghazal; Cravador, Alfredo

    2014-01-01

    cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-β-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-β-glucanase (QsGlu). Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), β-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work. Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes.

  10. European interlaboratory comparison of Schmallenberg virus (SBV) real-time RT-PCR detection in experimental and field samples: The method of extraction is critical for SBV RNA detection in semen.

    Science.gov (United States)

    Schulz, Claudia; van der Poel, Wim H M; Ponsart, Claire; Cay, Ann Brigitte; Steinbach, Falko; Zientara, Stéphan; Beer, Martin; Hoffmann, Bernd

    2015-07-01

    Molecular methods for the detection of Schmallenberg virus (SBV) RNA were rapidly developed after the emergence of this novel orthobunyavirus in Europe. The SBV epizootic wave has declined, but infectious SBV in SBV RNA-positive semen remains a possible risk for the distribution of SBV. However, the abilities of SBV molecular detection methods used at European laboratories have not yet been assessed, to our knowledge. The performances of extraction and real-time reverse transcription polymerase chain reaction (RT-qPCR) methods used at 27 German and 17 other European laboratories for SBV RNA detection in the matrices of whole blood, serum, tissue homogenate, RNA eluates, and bovine semen were evaluated in 2 interlaboratory trials with special emphasis on semen extraction methods. For reliable detection of viral genome in bovine semen samples, highly effective extraction methods are essential to cope with the potential inhibitory effects of semen components on PCR results. All methods used by the 44 laboratories were sufficiently robust to detect SBV RNA with high diagnostic sensitivity (100%) and specificity (95.8%) in all matrices, except semen. The trials demonstrated that the published recommended semen extraction methods (Hoffmann et al. 2013) and a combination of TRIzol LS with an alternative extraction kit have a considerably higher diagnostic sensitivity to detect SBV RNA in semen up to a detection limit of Cq ≤35 compared to other extraction methods used. A thorough validation of extraction methods with standardized semen batches is essential before their use for SBV RNA detection in bovine semen.

  11. Relative quantification of PIK3CA gene expression level in fine-needle aspiration biopsy thyroid specimens collected from patients with papillary thyroid carcinoma and non-toxic goitre by real-time RT-PCR

    Directory of Open Access Journals (Sweden)

    Wojciechowska-Durczyńska Katarzyna

    2010-08-01

    Full Text Available Abstract Background Recent studies have shown that the phosphatidylinositol 3-kinase (PI3K signaling pathway is important regulator of many cellular events, including apoptosis, proliferation and motility. PI3K pathway alterations (PIK3CA gene mutations and/or amplification have been observed in various human tumours. In the majority of diagnosed cases, mutations are localized in one of the three "hot spots" in the gene, responsible for coding catalytic subunit α of class I PI3K (PIK3CA. Mutations and amplification of PIK3CA gene are characteristic for thyroid cancer, as well. Methods The aim of our study was to examine a gene expression level of PIK3CA in fine-needle aspiration biopsy (FNAB thyroid specimens in two types of thyroid lesions, papillary thyroid carcinoma (PTC and non-toxic goitre (NTG. Following conventional cytological examination, 42 thyroid FNAB specimens, received from patients with PTC (n = 20 and NTG (n = 22, were quantitatively evaluated regarding PIK3CA expression level by real-time PCR in the ABI PRISM® 7500 Sequence Detection System. Results Significantly higher expression level (RQ of PIK3CA in PTC group has been noted in comparison with NTG group (p Conclusion These observations may suggest role of PIK3CA alterations in PTC carcinogenesis.

  12. Simultaneous detection and differentiation by multiplex real time RT-PCR of highly pathogenic avian influenza subtype H5N1 classic (clade 2.2.1 proper and escape mutant (clade 2.2.1 variant lineages in Egypt

    Directory of Open Access Journals (Sweden)

    Arafa Abdel-Satar

    2010-10-01

    Full Text Available Abstract Background The endemic status of highly pathogenic avian influenza virus (HPAIV of subtype H5N1 in Egypt continues to devastate the local poultry industry and poses a permanent threat for human health. Several genetically and antigenically distinct H5N1 lineages co-circulate in Egypt: Strains of clade 2.2.1 proper replicate mainly in backyard birds causing the bulk of human infections, while a variant lineage within 2.2.1 (2.2.1v appears to be perpetuated mainly in commercial poultry farms in Egypt. Viruses of the 2.2.1v lineage represent drift variants escaping from conventional vaccine-induced immunity and some of these strains also escaped detection by commercial real time reverse transcriptase PCR (RT-qPCR protocols due to mismatches in the primers/probe binding sites. Results We developed therefore a versatile, sensitive and lineage-specific multiplex RT-qPCR for detection and typing of H5N1 viruses in Egypt. Analytical characterization was carried out using 50 Egyptian HPAIV H5N1 strains isolated since 2006 and 45 other avian influenza viruses (AIV. A detection limit of 400 cRNA copies per ml sample matrix was found. Higher diagnostic sensitivity of the multiplex assay in comparison to other generic H5 or M-gene based RT-qPCR assays were found by examination of 63 swab samples from experimentally infected chickens and 50 AIV-positive swab samples from different host species in the field in Egypt. Conclusions The new multiplex RT-qPCR assay could be useful for rapid high-throughput monitoring for the presence of HPAIV H5N1 in commercial poultry in Egypt. It may also aid in prospective epidemiological studies to further delineate and better control spread of HPAIV H5N1 in Egypt.

  13. Effects of biosurfactant produced by Lactobacillus casei on gtfB, gtfC, and ftf gene expression level in S. mutans by real-time RT-PCR

    Directory of Open Access Journals (Sweden)

    Omid Savabi

    2014-01-01

    Full Text Available Background: The Streptococci are the pioneer strains in plaque formation and Streptococcus mutans are the main etiological agent of dental plaque and caries. In general, biofilm formation is a step-wise process, which begins by adhesion of planktonic cells to the surfaces. Evidences show that expression of glucosyltransferase B and C (gtfB and gtfC and fructosyltransferase (ftf genes play critical role in initial adhesion of S. mutans to the tooth surface which results in formation of dental plaques and consequently caries and other periodontal disease. Materials and Methods: The aim of this study was to determine the effect of biosurfactants produced by a probiotic strain; Lactobacillus casei (ATCC39392 on gene expression profile of gftB/C and tft of S. mutans (ATCC35668 using quantitative real-time PCR. Results: The application of the prepared biosurfactant caused dramatic down regulation of all the three genes under study. The reduction in gene expression was statistically highly significant (for gtfB, P > 0.0002; for gtfC, P > 0.0063, and for ftf, P > 0.0057. Conclusion: Considerable downregulation of all three genes in the presence of the prepared biosurfactant comparing to untreated controls is indicative of successful inhibition of influential genes in bacterial adhesion phenomena. In view of the importance of glucosyltransferase gene products for S.mutans attachment to the tooth surface which is the initial important step in biofilm production and dental caries, further research in this field may lead to an applicable alternative for successful with least adverse side effects in dental caries prevention.

  14. Low cost real time interactive analysis system

    Science.gov (United States)

    Stetina, F.

    1988-01-01

    Efforts continue to develop a low cost real time interactive analysis system for the reception of satellite data. A multi-purpose ingest hardware software frame formatter was demonstrated for GOES and TIROS data and work is proceeding on extending the capability to receive GMS data. A similar system was proposed as an archival and analysis system for use with INSAT data and studies are underway to modify the system to receive the planned SeaWiFS (ocean color) data. This system was proposed as the core of a number of international programs in support of U.S. AID activities. Systems delivered or nearing final testing are listed.

  15. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, Johan Frederik; Pierik, A.

    2012-01-01

    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the

  16. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, J.F.; Pierik, A.

    2012-01-01

    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the adva

  17. Detection of Runx2 mRNA expression using relatively real-time RT-PCR in papillary thyroid carcinoma%相对定量real time RT-PCR检测Runx2 mRNA在甲状腺乳头状癌中的表达

    Institute of Scientific and Technical Information of China (English)

    龚婷; 王家东; 钱敏飞; 周雅琪

    2013-01-01

    目的:相对定量检测Runx2 mRNA在甲状腺乳头状癌和腺瘤中的表达,并探讨其在甲状腺乳头状癌的发生发展及与钙化的意义.方法:相对定量real time RT PCR检测14例甲状腺乳头状癌和14例甲状腺腺瘤中Runx2mRNA的表达.结果:癌组和腺瘤组的△CT值分别为2.395±0.302和5.028±1.179,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01);癌组和腺瘤组的2-△△CT分别为7.826±5.004和1,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01).癌组和腺瘤组内以钙化分组△CT结果示P>0.05,组间差异均无统计学意义.癌组按肿瘤大小<1 cm和≥1 cm分组,△CT值分别为2.629±0.300和2.212±0.124,P<0.05;2-△△CT值分别为167.33±33.823和221.69±18.843,P<0.01.癌组和腺瘤组以及癌组内钙化分组TSH水平比较均P>0.05.结论:Runx2在甲状腺乳头状癌中的表达高,并与癌大小有关,在较大的癌中表达较高.Runx2与微钙化的关系,可能与甲状腺乳头状癌内钙化灶的产生及癌的发生发展有关,在其他的恶性肿瘤(如乳腺癌、前列腺癌、骨肉瘤)也有相关研究.%Objective:To relatively detect the Runx2 mRNA expression in papillary thyroid carcinoma(PTC) and thyroid adenoma, then to investigate the role of Runx2 in the development and progression in PTC and the relationship with the micro calcification in PTC. Method:The expression of Runx2 mRNA in 14 samples of PTC and 14 samples of thyroid adenoma was examined by relatively real-time RT-PCR. Result;The ACT value of the carcinoma group and adenoma group was 2. 395 + 0. 302 and 5. 028±1. 179 respectively(P<0. 01). The 2-△△CT value of the carcinoma group and adenoma group was 7. 826 + 5. 004 and 1 respectively(P<0. 01). The carcinoma group was divided into two groups by calcification and there was no statistical difference(P>0. 05). and the adenoma group as well. The carcinoma group was divided into

  18. Primary synovial sarcoma of the heart: a cytogenetic and molecular genetic analysis combining RT-PCR and COBRA-FISH of a case with a complex karyotype.

    Science.gov (United States)

    Hazelbag, Hans Martin; Szuhai, Károly; Tanke, Hans J; Rosenberg, Carla; Hogendoorn, Pancras C W

    2004-11-01

    Synovial sarcomas usually occur in the soft tissues of the extremities of adolescents and middle-aged patients, in the vicinity of large joints. We present a patient with a synovial sarcoma of the left atrium and ventricle, which is an extremely rare location. Diagnosis was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR), showing the t(X;18) fusion transcript. With a multicolor COmbined Binary RAtio labeling Fluorescence In Situ Hybridization (COBRA-FISH) technique, a complex karyotype evolved with identification of derivative chromosomes with multiplex rearrangements. This underscores the importance of molecular analysis of spindle cell tumors in unusual locations. Moreover, it shows that the presumed diagnostic translocation t(X;18) can be embedded in a sequence of other chromosomal rearrangements of which the function is as yet unknown.

  19. 鸭Ⅰ型肝炎病毒和番鸭细小病毒二重荧光定量RT-PCR方法的建立%Development of duplex real-time RT-PCR assay for detection of duck hepatitis virus type Ⅰ and muscovy duck parvovirus

    Institute of Scientific and Technical Information of China (English)

    谢丽基; 谢芝勋; 邓显文; 谢志勤; 庞耀珊; 范晴; 刘加波

    2013-01-01

    A duplex real-time reverse polymerase chain reaction (drRT-PCR) assay was developed and optimized to simultaneously detect duck hepatitis virus type Ⅰ and muscovy duck parvovirus in one reaction.Two sets of specific oligonucleotide primers for duck hepatitis virus type Ⅰ and muscovy duck parvovirus,along with two TaqMan probes specific for each virus were used in the assay.This drRT-PCR assay was found to be specific and to be able to detect and differentiate duck hepatitis virus type Ⅰ and muscovy duck parvovirus,and no positive results were observed when nucleic acid from duck circovirus,gosling plague virus,duck parvovirus,duck plague virus and avian influenza virus were used as drRT-PCR templates.The sensitivity of this drRT-PCR assay was 200 template copies for duck hepatitis virus type Ⅰ and muscovy duck parvovirus.This drRT-PCR assay is a quick,sensitive,and specific test for detection of duck hepatitis virus type Ⅰ and muscovy duck parvovirus,and will be useful for the control of these viruses in ducks.%根据基因库中鸭Ⅰ型肝炎病毒和番鸭细小病毒的基因序列,设计2对特异性引物和2条用不同荧光基团标记的TaqMan探针.对反应条件和试剂浓度进行优化,建立能够同时检测鸭Ⅰ型肝炎病毒和番鸭细小病毒的二重荧光定量RT-PCR方法.该方法敏感性好,对鸭Ⅰ型肝炎病毒和番鸭细小病毒的检测敏感性均达到200个模板拷贝数;该方法特异性强,对鸭圆环病毒、鹅细小病毒、鸭副黏病毒、鸭瘟病毒和禽流感病毒等病原体的检测全为阴性.本研究建立的鸭Ⅰ型肝炎病毒和番鸭细小病毒的二重荧光定量RT-PCR方法,具有快速、敏感、特异、定量和重复性好等优点,可用于临床上鸭Ⅰ型肝炎病毒和番鸭细小病毒感染的检测.

  20. 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

    Science.gov (United States)

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

  1. Quantification of transcript levels with quantitative RT-PCR.

    Science.gov (United States)

    Carleton, Karen L

    2011-01-01

    Differential gene expression is a key factor driving phenotypic divergence. Determining when and where gene expression has diverged between organisms requires a quantitative method. While large-scale approaches such as microarrays or high-throughput mRNA sequencing can identify candidates, quantitative RT-PCR is the definitive method for confirming gene expression differences. Here, we describe the steps for performing qRT-PCR including extracting total RNA, reverse-transcribing it to make a pool of cDNA, and then quantifying relative expression of a few candidate genes using real-time or quantitative PCR.

  2. 猪瘟病毒Taqman实时定量RT-PCR检测方法的建立和临床应用%Development and clinical application of Taqman real-time RT-PCR assay for detection of classical swine fever virus

    Institute of Scientific and Technical Information of China (English)

    毛立; 李文良; 李彬; 江杰元

    2012-01-01

    According to the conservative sequences located on the 5' untranslated region (5'-UTR) of classical swine fever virus(CSFV) ,a pair of specific primers and Taqman probe were designed and synthesized respectively, and a Taqman real-time fluorescent quantitative reserve-transcribed polymerase chain reaction (real-lime RT-PCR) for detecting the CSFV was established in this study. Test results showed that the method had a detection limit of 10 copies of target RNA per reaction, and there was a good linear relationship between Ct value and copy numbers in diluted samples. The variation between batches was less than 1% . The RNA of porcine reproductive and respiratory syndrome virus,bovine viral diarrhea virus were detected by the Taqman RT-PCR,and the results were all negative. The CSFV-positive rate was 71. 9% in 192 samples collected from Jiangsu and Xinjiang areas. Real-time RT-PCR detection showed that the different organs of swine including hearts, lungs, livers, kidneys, brains, spleens,lymph nodes and ascites were CSFV-positive, indicating thai the method were more sensitive and effective than traditional RT-PCR.%根据猪瘟病毒5’非编码区(5’-UTR)设计特异性引物和Taqman探针,建立Taqman实时定量RT-PCR检测猪瘟病毒法.检测结果显示,该方法的灵敏度为1μl 10拷贝,在病毒拷贝数为1μl 108~101时,循环数(Ct)值与拷贝数对数呈现较好的线性关系,且重复性好,批间变异系数小于1%.用该方法检测猪繁殖与呼吸综合征病毒、牛病毒性腹泻病毒,结果均为阴性.用该方法检测采集自江苏和新疆的192份组织和血清样品,猪瘟病毒阳性率为71.9%;检测感染猪的不同脏器,发现在心、肺、肝、肾、脑、脾脏、淋巴结、腹水中均可以检测到猪瘟病毒,与常规RT-PCR方法相比,该方法敏感性更高.该方法的建立为猪瘟病毒的流行病学调查和定量提供了有效手段.

  3. On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

    Science.gov (United States)

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

    2015-01-01

    NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate 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 and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of

  4. Development of SYBR Green Ⅰ real-time RT-PCR to detect SOCS1 in guinea pigs%豚鼠细胞因子信号抑制蛋白1基因SYBR Green Ⅰ荧光定量PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    许微微; 孙丽娟; 张坤; 王铁成; 赵永坤; 黄耕; 冯娜; 杨松涛; 高玉伟

    2013-01-01

    目的 建立豚鼠细胞因子信号抑制蛋白(SOCS1) SYBR GreenⅠ荧光定量PCR方法. 方法 根据GenBank上公布的豚鼠全基因序列,设计SOCS1全长引物,并在其保守区设计Real-time PCR扩增引物.对SOCS1全长PCR扩增,构建pEASY-Blunt-SOCS1标准质粒.利用10倍梯度稀释标准质粒作为模版,建立SYBR Green Ⅰ荧光定量PCR检测方法. 结果 成功建立了标准曲线,相关系数为0.996,扩增效率为103.7%,无非特异性产物和引物二聚体产生,初步应用结果显著. 结论 本实验建立的豚鼠细胞因子信号抑制蛋白SYBR Green Ⅰ荧光定量PCR方法可用于细胞因子抑制信号蛋白变化的快速定量检测,能够为禽流感H5N1的研究提供一定的技术支持.%Objective In order to study the expression of SOCS in guinea pigs infected with the avian influenza virus (AIV),a rapid assay using SYBR Green Ⅰ real-time RT-PCR was developed to detect SOCS1 in guinea pigs Methods The sequence of the SOCS1 gene of guinea pigs was obtained from GenBank and analyzed using the tool MegAlign in DNAStar software.A full-length primer was designed along with a primer for PCR amplification of its conserved region.The standard plasmid was diluted 10-fold.Results Results indicated that the correlation coefficient for the standard curve was 0.996 and the efficiency of amplification was 103.7%.The standard plasmid was constructed to verify the accuracy of real-time PCR.Non-specific production and primer dimerization were not noted.In addition,the technique yielded significant results in its initial use.Conclusion The SYBR Green Ⅰ RT-PCR technique developed in this study can rapidly detect SOCS and it can facilitate study of the AIV.

  5. Detección y caracterización del virus de bronquitis infecciosa aviaria en Chile mediante RT-PCR y análisis secuencial Detection and characterization of infectious bronchitis virus in Chile by RT-PCR and sequence analysis

    Directory of Open Access Journals (Sweden)

    J C Lopez

    2006-01-01

    Full Text Available Una técnica de reacción en cadena de la polimerasa transcriptasa reversa (RT-PCR junto a una secuenciación fue usada para detectar y caracterizar genéticamente virus diferentes de bronquitis infecciosa aviar (VBIA aislados en Chile. El procedimiento de RT-PCR incluyó el uso de los partidores NT1 y NT2, los cuales se localizaron cerca del término N del gen S1 y cubrieron la región hipervariable. La secuencia amplificada fue alineada y analizada con el programa computacional DNAman, y comparada con secuencias reportadas en GenBank. El nivel de detección de la técnica de RT-PCR fue equivalente al aislamiento viral en huevos cuando se usaron directamente tejidos, pero el ensayo fue más sensitivo cuando fue usado para detectar virus almacenados en fluido alantoideo. Los amplificados de todos los aislados históricos de Chile fueron idénticos en tamaño (193pb y exhibieron entre ellos, al analizar la secuencia una similitud del 71 al 96%. Estos aislados mostraron entre 68 y 97% de similitud con cepas de Estados Unidos, Europa, Asia, Nueva Zelandia y Australia.A reverse transcriptase-polymerase chain reaction (RT-PCR assay, coupled with sequencing, was used to detect and genetically characterize different infectious bronchitis virus (IBV isolates in Chile. The RT-PCR procedure included the use of the primers NT1 and NT2 that were located close to the N-terminus of the S1 gene and bracketed the hypervariable region, and the amplified sequences were aligned and analyzed with DNAman software, and compared with sequences from GenBank. The level of detection of the RTPCR assay was equivalent to virus isolation in eggs when testing tissues directly, but the assay was more sensitive when used to detect virus stored in allantoic fluid. The amplimers from all historical Chilean isolates were identical in size (193 bp and exhibited 71-96% similarity on sequence analysis. These isolates showed between 68-97% similarity to strains from North America

  6. Analysis of ORF 1 in European porcine reproductive and respiratory syndrome virus by long RT-PCR and restriction fragment length polymorphism (RFLP) analysis

    DEFF Research Database (Denmark)

    Nielsen, H. S.; Storgaard, Torben; Oleksiewicz, M.B.

    2000-01-01

    A rapid method was developed for partial characterization of the replicase-encoding open reading frame 1 (ORF 1) of porcine reproductive and respiratory syndrome virus (PRRSV). It comprised long RT-PCR amplification of 11.1 kb (94%) of ORF 1, followed by restriction fragment length polymorphism...

  7. 实时定量RT-PCR检测肝癌miRNA表达中内参的选择%Selection of optimal internal controls for miRNA expression in human hepatocellular carcinoma using real-time quantitative RT-PCR

    Institute of Scientific and Technical Information of China (English)

    党裔武; 陈罡; 容敏华

    2012-01-01

    目的 探讨肝癌(hepatocellular carcinoma,HCC)组织采用实时定量RT-PCR(RT-qPCR)技术检测miRNA表达最佳内参的选择.方法 采用实时RT-qPCR技术检测7个内参RNU6B、RNU48、RNU66、RNU44、RNU43、5s rRNA和18s rRNA在70例HCC及相应癌旁肝组织中的表达.并使用NormFinder及geNorm软件分析最优化的内参.结果 7个内参表达结果存在差异.NormFinder及geNorm软件均显示RNU6B+RNU48组合为肝组织的最佳内参.结论 使用实时定量RT-qPCR研究目标miRNA相对定量表达时,为保证数据的精确性和客观性,在实验前对内参进行慎重的筛选是必不可少的.NormFinder及geNorm软件可用于各种实验因素下适宜内参的选择.%Purpose To investigate the selection of optimal internal controls for miRNA expression in human hepatocellular carcinoma ( HCC ) using real-time quantitative RT-PCR ( RT-qPCR ). Methods Expression of 7 controls ( RNU6B, RNU48, RNU66, RNU44, RNU43 , 5s rRNA and 18s rRNA ) was detected with real-time RT-qPCR in 70 cases of HCC and their adjacent non-cancerous liver tissues. The optimal internal controls were selected by NormFinder and geNorm softwares. Results The genes studied displayed a wide expression range with different Cq values. Both NormFinder and geNorm indicated that the optimal internal controls for liver tissue were the combination of RNU6B and RNU48. Conclusions In order to ensure the accuracy and objectivity of normalized data in RT-qPCR studies adopting relative miRNA quantification, careful selection and validation of endogenous controls are advocated as a mandatory step. NormFinder and GeNorm softwares can be used to screen out the most stable controls under different experimental conditions.

  8. Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

    Science.gov (United States)

    Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

    2017-07-25

    Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an

  9. 实时荧光RT-PCR检测不同标本中甲型H1N1流感病毒核酸的临床意义%Clinical significance of fluorescent real-time RT-PCR in the detection of influenza A virus (H1N1) nucleic acid in different types of specimens

    Institute of Scientific and Technical Information of China (English)

    陈双峰; 陈海英; 张颖新; 郑丽丽

    2011-01-01

    Objective To explore the relationship of influenza A virus ( H1N1) nucleic acid with rapid diagnosis of influenza A ( H1N1) and the progress of disease course by the detection of viral nucleic acid in different specimens from patients with influenza A ( H1N1). Methods Fluorescent real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect influenza A virus ( H1N1) nucleic acid in throat swabs, plasma and secretion of lower respiratory tract from 118 patients with influenza A ( H1N1) 48 hours after onset and (or) during convalescence respectively. Results Viral nucleic acid was detected in throat swabs from all 118 patients 48 hours after onset,secretion of lower respiratory tract from 23 severe cases and plasma from 15 severe cases. Coriclusion Fluorescent real -time RT-PCR could be used for the rapid detection of influenza A virus ( H1N1) infection. Influenza A virus (H1N1) nucleic acid,when be detected in plasma,may be the indication of the severe cases.%目的 通过检测甲型H1N1流感感染患者不同标本中甲型H1N1流感病毒核酸,了解其对甲型H1N1流感的快速诊断及病程进展的关系.方法 118例甲型H1N1流感患者,采用实时荧光逆转录聚合酶链反应法(RT-PCR)分别检测发病48 h后和(或)恢复期咽拭子、血浆及下呼吸道分泌物中甲型H1N1流感病毒核酸.结果 118例甲型H1N1流感患者发病48 h后咽拭子甲型H1N1流感病毒核酸阳性;23例(23/40)重症患者下呼吸道分泌物中甲型H1N1流感病毒核酸阳性;15例(15/40)重症患者血浆中检测到甲型H1N1流感病毒核酸.结论 实时荧光RT-PCR方法可以快速检测甲型H1N1流感病毒感染,血浆中甲型H1N1流感病毒核酸阳性可能是重症患者的标志.

  10. Real-Time Principal-Component Analysis

    Science.gov (United States)

    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. 急性早幼粒细胞白血病PML-RARα融合基因实时定量RT-PCR标化法和常规法计算的比较%Comparisons of conventional and normalized calculation of quantitative real-time RT-PCR detecting PML-RARα fusion gene in patients with acute promyelocytic leukemia

    Institute of Scientific and Technical Information of China (English)

    胡炯; 高晓东; 刘元北; 朱勇梅; 李军民; 沈志祥

    2008-01-01

    目的 改进实时定量RT-PCR的检测计算方法,提高定量RT-PCR在急性早幼粒细胞白血病(APL)微量残留病变监测的临床应用价值.方法 采用实时定量RT-PCR和常规定性RT-PCR检测31例APL患者在治疗前后融合基因PML-RARα的表达水平,对两种计算方法进行比较:常规按照患者治疗前后自身比较计算(常规法)和治疗前标准化基线水平计算(标化法).结果31例患者采用两种定量计算方法结果近似,标化法计算结果示患者在治疗缓解、巩固治疗和维持治疗期间融合基因PML-RARα转录本对数下降值分别为(2.0±1.9)、(4.9±1.4)和(5.7±0.1),常规法计算结果为(1.9±1.9)、(4.8±1.3)和(5.7±0.4).在维持治疗阶段标化法定量RT-PCR的结果显示患者个体差异更小.按照标化法计算结果,次要分子生物反应(对数值≥3)和主要分子生物反应(对数值≥5)标准仍然适用.结论 采用标化法计算实时定量RT-PCR结果能较好的对APL患者的微量残留病变进行监测,一定程度上降低了患者个体差异对检测结果的影响,同时适用于治疗前标本缺如的患者.%Objective To optimize the calculation of quantitative real time RT-PCR (Q-RT-PCR) of PML-RARα in patients with acute promyelocytic leukemia (APL) for molecular monitoring of minimal residual disease(MRD). Methods By using both regular reverse transcription polymerase chain reaction (RT-PCR) and Q-RT-PCR, the expression levels of PML-RARα transcripts were measured before and after treatment. The conventional Q-RT-PCR calculation was directly compared the post-treatment transcript level with the respective pre-treatment one (DoseN) in the individual patient while the standardized calculation was based on the calculation of standardized pre-treatment DoseN of all patients. Results In 181 samples from 31 patients, the results of log-reduction of PML-RARα after induction, at the end of consolidation and during maintenance by conventional

  12. CRANS - CONFIGURABLE REAL-TIME ANALYSIS SYSTEM

    Science.gov (United States)

    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

  13. 应用含内参的多重实时荧光RT-PCR方法快速检测登革病毒和基孔肯雅病毒%Rapid detection of Dengue virus and Chikungunya virus by multiplex real-time RT-PCR assay with an internal control

    Institute of Scientific and Technical Information of China (English)

    郑夔; 丁国允; 周惠琼; 谢雪妹; 李小波; 师永霞; 苏锦坤; 黄吉城

    2013-01-01

    目的 建立一种登革病毒、基孔肯雅病毒并含人类基因内参检测的多重实时荧光RT-PCR方法,能在同一反应管内同时检测目前发现的所有来源的登革病毒或基孔肯雅病毒.方法 针对登革病毒3′端非编码区和基孔肯雅病毒结构蛋白E2-6K-E1区以及人体各类组织细胞中均能稳定表达的RNAse P基因,设计了3套特异性引物和探针,建立了1套能同时检测登革病毒、基孔肯雅病毒及含有人类基因检测内参的多重实时荧光RT-PCR方法,对其灵敏性和特异性进行了验证,并对临床发热病人标本进行了应用评估.结果 该方法对检测体外转录合成的登革病毒和基孔肯雅病毒RNA的灵敏性可达最低每个反应10~100拷贝,对检测登革1型病毒和基孔肯雅病毒的灵敏性分别可达最低每个反应0.1 TCID50/mL和1 TCID50/mL.用20株登革病毒、4株基孔肯雅病毒和日本脑炎病毒、西尼罗病毒、黄热病毒、盖塔病毒、辛德毕斯病毒各1株进行检测,方法的特异性均为100%.方法应用于189份发热病人血清标本检测,可准确地鉴定出其中登革病毒或基孔肯雅病毒核酸阳性的标本,且所有血清标本均能被内参引物和探针有效地扩增和杂交.结论 本研究建立了一种高灵敏性、高特异性且含人类基因内参检测的登革病毒和基孔肯雅病毒多重实时荧光RT-PCR检测方法,可作为登革热或基孔肯雅热病人早期快速鉴别诊断的有效工具,也可用于蚊媒携带登革病毒或基孔肯雅病毒的高通量快速筛查.%The purpose was to establish a multiplex real-time RT-PCR assay for detection of Dengue virus and Chikun-gunya virus in one tube, which could detect all Dengue virus or Chikungunya virus strains from different origins . Based on sequences of 3 -UTR of Dengue virus , E2-6K-E1 region of Chikungunya virus's structural protein and RNAse P gene which stably expressed in all human organs , 3 pairs of

  14. A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

    Directory of Open Access Journals (Sweden)

    Saegerman Claude

    2008-08-01

    Full Text Available Abstract Background Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. Due to their genetic proximity, potential zoonotic transmission or animal reservoir can be hypothesized for noroviruses. RT-PCR has become the "gold standard" for the detection of noroviruses in faecal and environmental samples. With such samples, the control for inhibition of the reaction during amplification and detection is crucial to avoid false negative results, which might otherwise not be detected. The aim of the reported method is to detect, with a SYBR Green technology, a broad range of noroviruses with a control for inhibition. Results A SYBR Green real-time RT-PCR assay was developed making use of a foreign internal RNA control added in the same tube. This assay is able to detect human and bovine noroviruses belonging to genogroups I, II and III and to distinguish between norovirus and internal control amplicons using melting curve analysis. A 10-fold dilution of samples appears to be the method of choice to remove inhibition. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. Conclusion This SYBR Green real-time RT-PCR assay allows the detection of the most important human and bovine noroviruses in the same assay, and avoids false negative results making use of an internal control. Melting curves allow the discrimination between the internal control and norovirus amplicons. It gives preliminary information about the species of origin. The sensitivity of the developed assay is higher than conventional RT-PCR and a 10-fold dilution of samples showed a better efficiency and reproducibility to remove RT-PCR inhibition than addition of bovine serum albumin.

  15. TaqMan(R)实时定量RT-PCR检测G3型轮状病毒VP7基因方法的建立%Establishment of a TaqMan(R) real-time RT-PCR for quantitating VP7 genes of rotavirus serotype G3

    Institute of Scientific and Technical Information of China (English)

    王云瑾; 余黎; 胡广宏; 陈继军; 周旭

    2012-01-01

    Objective; To establish a TaqMan(R) real-time polymerase chain reaction for the detection and quantitative measurement of VP7 gene of G3 human rotaviruses. Methods: Primers and probe specific for the VP7 gene of G3 human rotaviruses were designed, and the TaqMan real-time RT-PCR protocol was established using in vitro transcription RNA standards. Results and Conclusion; This assay results in a rapid, sensitive and reproducible detection of VP7 gene of rotaviruses ranging from 107~101 copies· μpL-1, providing a useful tool for the detection and quantification of G3 rotaviruses.%目的:建立一种检测G3型轮状病毒VP7基因的TaqMan(R)实时定量RT-PCR方法.方法:设计轮状病毒VP7基因型特异性引物和探针,采用体外转录RNA为标准品,建立TaqMan(R)实时定量RT-PCR方法.结果与结论:本检测方法快速、灵敏且重复性好,可以对107~101 copies·μL-1轮状病毒VP7基因进行有效检测,为G3型轮状病毒的检测和定量提供了有用的工具.

  16. Rapid assay for H7N9 avian influenza virus genes by real-time fluorescence quantitative RT-PCR%实时荧光定量RT-PCR快速检测H7N9禽流感病毒

    Institute of Scientific and Technical Information of China (English)

    张舟听; 夏文英; 王忠发; 刘妙; 翁瑛瑛

    2014-01-01

    目的 构建一种灵敏、特异、快速的实时荧光定量RT-PCR反应体系用于临床H7N9禽流感疑似病例早期诊断及外环境H7N9病毒的监测.方法 以H7N9禽流感病毒H7基因和N9基因为靶基因设计引物以及TaqMan探针,并对引物、探针及反应条件进行优化,验证该反应体系检测的特异性、灵敏度、重复性和检测速度,然后与两种商品化试剂盒(试剂盒I和试剂盒Ⅱ)进行各项指标及184份现场样本作平行比对.结果 新建立的H7N9禽流感病毒实时荧光定量RT-PCR反应体系呈典型的扩增曲线、扩增时间约50 min,H7基因与N9基因的最低检出限分别为28拷贝/μL与41拷贝/μL.重复性测定显示,H7和N9检测Ct值的变异系数(CV)分别为0.17%~0.89%和0.33%~0.59%,扩增效率分别为0.9491和0.9713;与其他常见禽流感病毒或肠道病毒无明显交叉反应.184份现场可疑样本阳性检出率为5.43%(10/184),与试剂盒Ⅱ结果一致.结论 本研究建立的实时荧光定量RT-PCR反应体系具有检测速度快、灵敏度高、特异性强、重复性好、结果可靠等特点,可用于临床疑似病例的应急检测、早期诊断及养鸡场、活禽交易市场等外环境H7N9禽流感病毒监测.%Objective To develop a sensitive,specific and rapid reaction system of real-time fluorescence quantitative RT-PCR for early diagnosing clinical H7N9 avian influenza suspected patients and monitoring environmental pollutions.Methods Primers and TaqMan probes were designed according to the H7 and N9 genes of H7N9 avian influenza virus,then the primers,probes and reaction conditions were optimized,the specificity,sensitivity,repeatability and detection speed were verified.The RT-PCR method developed was compared with two commercial kits (Kit I and Kit Ⅱ)in every parameter and 184 field samples.Results The real-time fluorescence quantitative RT-PCR developed for detecting H7N9 avian influenza virus had typical

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

    Science.gov (United States)

    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...

  18. 茂名市首例甲型H1N1流感病例的实时荧光RT-PCR检测%Testing for the First Patient of Influenza A (H1N1) Virus by Real-time RT-PCR in Maoming City

    Institute of Scientific and Technical Information of China (English)

    廖国东; 陈家图; 罗光毅; 何展; 杨适乡; 胡乐

    2009-01-01

    [目的]对茂名市首例甲型H1N1流感病例进行病毒核酸检测,为甲型H1N1流感疫情的防控提供参考.[方法]采用实时荧光逆转录PCR法对采集的病人咽拭子标本进行甲型H1N1流感病毒核酸检测.[结果]共采集病人2份咽拭子标本,其中1份咽拭子标本的实时荧光RT-PCR检测结果显示H1及N1的RT-PCR反应体系扩增曲线有明显对数增长且Ct值分别为25.21(H1)和25.07(N1),为甲型H1N1流感病毒核酸阳性;另1份咽拭子标本则呈阴性.后采集第3份咽拭子标本连同第1分标本送广东省疾控中心复检,结果2份标本的甲型H1N1流感病毒核酸检测均为阳性.[结论]这名病人为福建省第2例甲型H1N1流感病毒感染者的密切接触者,由于及时采用灵敏度高,特异性强,检测时间短的实时荧光逆转录PCR方法检测,快速确诊了病例,为疫情的控制争取了时间,防止了2代病例的出现.%Objective To test the Virus nucleic acid for the first patient of Influenza A (HIN1) in Maoming City, and to provide the references for prevention and control of Influenza A (H1N1). Method The throat swab of patient was collected and tested by Real-time PCR. Result In 2 samples of throat swabs the 1st throat swab had the obvious logarithm increasing for H1 and N1 in reaction system of Real-time RT-PCR, the Ct numeric of H1 and N1 was 25.21 and 25.07, it was positive of Influenza A (H1N1); but the 2nd throat swab was negative. The 3rd throat swab was sent to Center for Disease Control and Prevention of Guangdong province with 1st throat swab, both the result of re-test of two throat swab showed positive of Influenza A (H1N1). Conclusion This patient was the close contact the 2nd infecter with Influenza A (H1N1) in Fujian province. This case was identified rapidly by Real-time RT-PCR which has high sensitivity and specificity. Meanwhile, the aggressive and preemptive measures have been taken in a timely manner, and the secondary cases have been

  19. 牛病毒性腹泻病毒和牛轮状病毒TaqMan二重实时荧光RT-PCR检测方法的建立%Detection of bovine viral diarrhea virus and bovine rotavirus by TaqMan based real-time RT-PCR

    Institute of Scientific and Technical Information of China (English)

    范晴; 谢芝勋; 刘加波; 庞耀珊; 邓显文; 谢志勤; 谢丽基; 彭宜

    2011-01-01

    根据牛病毒性腹泻病毒(BVDV)5′端非编码区和牛轮状病毒(BRV)VP6基因序列,设计特异性引物和探针。通过对引物和探针浓度、Mg2+浓度、dNTP浓度和Taq酶用量以及反应条件等因素的优化筛选,建立了能同时鉴别BVDV和BRV的二重荧光RT-PCR方法。该方法特异性好,与其他病原如CSFV、MB和IBRV不发生交叉反应;敏感性高,能够检测100个BVDV RNA和100个BRV RNA;稳定性好,批内重复和批间重复变异系数小;干扰性试验表明该方法能同时检测2个模板的不同浓度组合。本研究建立的二重荧光RT-PCR方法可用于BVDV和BRV检测,具有特异、敏感、快速、稳定等优点,是BVDV和BRV基础研究、流行病学调查和临床检测的良好工具。%Two pairs of primers and two TaqMan probes were designed and synthesized according to the conserved gene sequence of BVDV 5′ untrascription region and BRV VP6.The reaction parameters such as the concentration of two pair of primers,two probes and other conditions were optimized to develop a duplex real-time RT-PCR assay for rapid detection of BVDV and BRV.It was found that the specificity of this assay was high,and be able to detected BVDV and BRV without other any cross-reactions to CSFV,MB and IBRV.The detection limit of the real-time RT-PCR assay was 100 copies of BVDV viral RNA and BRV viral RNA,indicating a good sensitivity of the assay.The coefficients of variation were both low for the intra-assay and inter-assay tests respectively,indicating a good reliability.When different concentration of BVDV and BRV was mixed together,the result was without any interference.All the resuls indicate that this duplex real-time PCR assay is a specific,sensitive,rapid and reproducible method for detection of BVDV and BRV,and is could applied in fundamental research,clinical detection and epidemiological investigation of BVDV and BRV.

  20. Real-time Forensic Disaster Analysis

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Svingen, Terje; 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.

  2. Real-Time Quantification of 3 microRNAs of Drosophila melanogaster by RNA-Tailing and Primer-Extension qRT-PCR%RNA加尾和引物延伸法检测黑腹果蝇3种microRNA表达量

    Institute of Scientific and Technical Information of China (English)

    戚晓娴; 李晓梅; 陈永; 钟国华

    2011-01-01

    采用RNA加尾和引物延伸real time PCR法实时定量检测了黑腹果蝇Drosophila melanogaster的bantam、mir14、mir-2a共3种microRNA(miRNA)在各发育阶段组织中的相对表达情况,发现3种miRNA在不同发育时期的表达差异明显,其表达变化规律与文献报道基本一致,验证了该法的可靠性.这种"加尾和引物延伸"方法,仅需1~10 ng总RNA或等同物,检测范围可达7个数量级,为昆虫miRNA研究提供了可靠的定量检测方法.%The modified RNA-tailing and primer-extension qRT-PCR method was used to detect relative quantity of 3 microRNAs, bantam, mir-14 and mir-2a, in various developmental stages in Drosophila melanogaster. The results showed that the expression patterns of these miRNAs had the significant differences in different developmental stages,and the change dynamics of miRNAs relative quantities were consistent with the literatures which verified the reliability of this method. This method of RNA-tailing and primerextension had advantages of high sensitivity ,which only needed 1 to 10 ng total RNA or equivalents,and wide detection range with 107 grades, compared with other methods,which would provide a reliable method of quantification detection in insect miRNAs researches.

  3. Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

    Science.gov (United States)

    2016-09-07

    modification or cleavages of transcripts through various RNA editing mechanisms , including siRNA targeted mRNA cleavages. Currently, 5′-RACE (rapid ampli... transcription polymerase chain reaction (SLA-RT-PCR) approach to detect and verify the siRNA- mediated target mRNA cleavage sites by determining precise...Reverse Transcription kit (Life Technologies) in combination with an array of stem-loop RT primers. The 20 ml of RT reaction con- tained 50 ng of total RNA

  4. 实时定量荧光反转录-聚合酶链反应检测急性白血病AML1/ETO融合基因%Real-time RT-PCR for detection and quantification of AML1/ETO leukemia fusion gene

    Institute of Scientific and Technical Information of China (English)

    李斌; 喻镁佳; 梁洋; 李晓进; 胡杰; 陈琪; 李惠民

    2009-01-01

    Objective To set up real-time quantitative RT-PCR technique and measure leukemia fusion gene transcripts in patients with AML of FAB-M_2 subtype,and also to investigate the positive rate in patients and the relationship between the AMLI/ETO mRNA levels and the response rate after chemical therapy.Methods The plasmid containing the AMLI/ETO fusion gene sequences were constructed from myeloid cell lines Kasumi-1 (expressing AML1/ETO)to establish the standard curves,A TaqMan based realtime quantitative RT-PCR was performed to measure aberrant fusion gene transcripts in 45 samples of peripheral blood(PB) or bone marrow (BM) from 25 newly diagnosed patients with AML-M_2.All these 25 patients were diagnosed by the FCM(flow cytometry)and bone marrow molecular cytogenetics,and received the induce remission therapy with MA (Mitoxantrone+Ara-C).Results As a result,the AML1/ETO fusion gene transcripts were detected in 7(28%)out of 25 AML-M_2 patients (the ratios of AML1/ETO/ABL vary from 0.01 to 19.2),in which 5 patients were found t(8;21)(q22;q22).The transcript level of AML1/ETO fusion gene varied from the clinical situation of patients.These 7 patients with AML1/ETO fusion gene got complete remission(CR) after the first MA therapy,and the fusion gene reduced by 3 log in AML1/ETO/ABL.Only 11 patients got CR in 18 patients without AML1/ETO fusion gene.By following up these 7 patients with AML1/ ETO fusion gene kept persistent CR for 6 months.Conclusion It was concluded that real-time quantitative PCR is a reliable,innovative and promising technology with high sensitivity and speciality.It has potential clinical value for diagnosis,tumor typing,treatment selection,measuring the tumor load,monitoring fusion gene expression level and evaluating therapeutic strategy.It is worthy to apply in the clinical practice.%目的 建立实时定量荧光反转录-聚合酶链反应(RQ RT-PCR)的方法并用来检测急性髓系白血病(AML)-M_2:患者中 AML1/ETO融合基因的拷贝

  5. Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR.

    Science.gov (United States)

    Wei, Libin; Miao, Hongmei; Zhao, Ruihong; Han, Xiuhua; Zhang, Tide; Zhang, Haiyang

    2013-03-01

    Sesame (Sesamum indicum L.) is an ancient and important oilseed crop. However, few sesame reference genes have been selected for quantitative real-time PCR until now. Screening and validating reference genes is a requisite for gene expression normalization in sesame functional genomics research. In this study, ten candidate reference genes, i.e., SiACT, SiUBQ6, SiTUB, Si18S rRNA, SiEF1α, SiCYP, SiHistone, SiDNAJ, SiAPT and SiGAPDH, were chosen and examined systematically in 32 sesame samples. Three qRT-PCR analysis methods, i.e., geNorm, NormFinder and BestKeeper, were evaluated systematically. Results indicated that all ten candidate reference genes could be used as reference genes in sesame. SiUBQ6 and SiAPT were the optimal reference genes for sesame plant development; SiTUB was suitable for sesame vegetative tissue development, SiDNAJ for pathogen treatment, SiHistone for abiotic stress, SiUBQ6 for bud development and SiACT for seed germination. As for hormone treatment and seed development, SiHistone, SiCYP, SiDNAJ or SiUBQ6, as well as SiACT, SiDNAJ, SiTUB or SiAPT, could be used as reference gene, respectively. To illustrate the suitability of these reference genes, we analyzed the expression variation of three functional sesame genes of SiSS, SiLEA and SiGH in different organs using the optimal qRT-PCR system for the first time. The stability levels of optimal and worst reference genes screened for seed development, anther sterility and plant development were validated in the qRT-PCR normalization. Our results provided a reference gene application guideline for sesame gene expression characterization using qRT-PCR system.

  6. Genetic grouping of avian infectious bronchitis virus isolated in Brazil based on RT-PCR/RFLP analysis of the S1 gene Agrupamento genético de isolados do vírus da bronquite infecciosa das aves no Brasil com base na análise do gene S1 por RT-PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Maria de Fátima S. Montassier

    2008-03-01

    Full Text Available Twelve Brazilian isolates and one reference vaccine strain of avian infectious bronchitis virus (IBV were propagated in embryonating chicken eggs. The entire S1 glycoprotein gene of these viruses was analysed by reverse-transcriptase-polymerase chain reaction and restriction fragment length polymorphism (RT-PCR-RFLP, using the restriction enzymes HaeIII, XcmI and BstyI. The RFLP patterns led to the classification of these isolates into five distinct genotypes: A, B, C, D and Massachusetts. Five of twelve isolates were grouped in Massachusetts genotype and the remaining seven viruses were classified into four distinct genotypes: A (2, B (2, C (2 or D (1. Such genotyping classification agreed with previous immunological analysis for most of these viruses, highlighting the occurrence of a relevant variability among the IBV strains that are circulating in Brazilian commercial poultry flocks.Doze isolados de campo do Brasil e uma estirpe de referência vacinal do vírus da bronquite infecciosa das aves (VBI foram propagadas em ovos embrionados SPF. O gene S1 dessas amostras foi analisado por RT-PCR seguido de RFLP, empregando-se as enzimas de restrição HaeIII, XcmI e BstyI. Observou-se a existência de cinco genotipos diferentes: M (Massachusetts, A , B, C e D. Cinco dos doze isolados de campo do VBI foram classificados no genótipo Massachusetts e os sete vírus restantes foram classificados em quatro genotipos diferentes; A (2, B (2, C (2 ou D (1. Os resultados desta genotipagem concordam com os dados obtidos na análise imunológica previamente realizada para a maior parte destes vírus, destacando a ocorrência de uma variabilidade marcante entre os isolados do VBI que estão circulando nas granjas avícolas comerciais do Brasil.

  7. Sentiment analysis on students’ real-time feedback

    OpenAIRE

    Altrabsheh, Nabeela

    2016-01-01

    Previous literature identifies that students’ real-time feedback is important in the learning process. There are numerous studies that have collected students’ feedback in real time. However, they include several limitations of which the most important is analysing the feedback. In this thesis, we address these limitations by proposing a system that will automatically analyse students’ feedback in real time and present the analysis results to the lecturer. To create such a system, we propose ...

  8. Evaluation of the reference genes for expression analysis using quantitative real-time polymerase chain reaction in the green peach aphid, Myzus persicae.

    Science.gov (United States)

    Kang, Zhi-Wei; Liu, Fang-Hua; Tian, Hong-Gang; Zhang, Meng; Guo, Shan-Shan; Liu, Tong-Xian

    2017-04-01

    The green peach aphid, Myzus persicae Sulzer (Hemiptera, Aphididae), is an important cosmopolitan pest. Real time qRT-PCR has been used for target gene expression analysis on M. persicae. Using real time qRT-PCR, the expression levels are normalized on the basis of the reliable reference genes. However, to date, the stability of available reference genes has been insufficient. In this study, we evaluated nine candidate reference genes from M. persicae under diverse experimental conditions. The tested candidate genes were comprehensively ranked based on five alternative methods (RefFinder, geNorm, Normfinder, BestKeeper and the comparative ΔCt method). 18s, Actin and ribosomal protein L27 (L27) were recommended as the most stable reference genes for M. persicae, whereas ribosomal protein L27 (L27) was found to be the least stable reference genes for abiotic studies (photoperiod, temperature and insecticide susceptibility). Our finding not only sheds light on establishing an accurate and reliable normalization of real time qRT-PCR data in M. persicae but also lays a solid foundation for further studies of M. persicae involving RNA interference and functional gene research.

  9. Establishment of a Real-time Fluorescent Quantitative RT-PCR Assay for Detection of Porcine IFN-β and IRF-3 genes%猪IFN-β和IRF-3基因实时荧光定量RT-PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    王蕊; 张改平; 乔松林; 刘永晖; 蒋志政; 万博; 鲍登克; 王爱萍

    2012-01-01

    为建立猪IFN-β及IRF-3基因实时荧光定量PCR检测方法,依据GenBank中IFN-β和IRF-3基因的保守序列,设计并合成各自特异性引物,并以β-actin为内参基因,采用SYBRGreen-Ⅰ为染料,建立实时荧光定量检测方法.提取猪肺泡巨噬细胞总RNA,经反转录得cDNA,用特异性引物经PCR扩增得到IFN-β和IRF-3基因,将其克隆至pMD-19T载体,经测序鉴定后得重组质粒,依次10倍稀释做为标准品,建立标准曲线及溶解曲线.结果表明,β3-actin基因、IFN-β基因和IRF-3基因标准曲线线性关系较好,R2≥0.997;溶解曲线为特异性单峰,无非特异性扩增,检测下限可达100个拷贝/μL.建立的猪IFN-β和IRF-3基因实时荧光定量RT-PCR检测方法,特异性强、重复性好,为从分子水平上研究猪的免疫应答奠定了基础.%To establish real-time fluorescent quantitative RT-PCR assays for detecting porcine IFN-β and IRF-3, sevral specific primer pairs were designed according to the porcine's IFN-β and IRF-3 gene sequences available in GenBank,and the porcine β-actin gene was used as an internal gene control. The total RNA was extracted from porcine alveolar macrophages. The reverse transcriptase PCR was used to obtain the first strand cDNA. Fragments for IFN-β and IRF-3 were amplified by PCR from the synthesized cDNA using the designed specific primers. The PCR products were purified and cloned into pMD-19T vector. The positive recombined plasmids were serially diluted and used as a standard. The standard and melting curve was analyzed. The results showed that the Ct value of β actin ,IFN-β and IRF-3 genes had a good linear relationship (R2≥ 0. 997) and the melting curve showed a single peak. The established real-time PCR methods can detect 100 copies of IFN-β and IRF-3 mRNA. The developed real-time PCR using SYBR Green I dye had high sensitivity,sepcifity and reproductivity.and could used as an effective tool for detection and quantification of IFN

  10. Comparative analysis of quantitative RT-PCR and conventional RT-PCR testing for hand, foot, and mouth disease%实时荧光RT-PCR与普通RT-PCR检测手足口病病原体的比较分析

    Institute of Scientific and Technical Information of China (English)

    许玉玲; 黄学勇; 卫海燕; 陈豪敏; 许汴利

    2011-01-01

    目的 对比分析实时荧光RT-PCR和普通RT-PCR方法在检测手足口病病原体的差别. 方法 选择临床诊断为手足口病患者粪便256份,提取RNA,用实时荧光RT-PCR和普通RT-PCR检测手足口病肠道病毒71型(EV71)、柯萨奇病毒A组16型(CoxA 16)和总肠道病毒(PE),对实验结果进行统计学分析. 结果 实时荧光RTPCR和普通RT-PCR检测手足口病患者粪便标本,CoxA 16结果差异有统计学意义(P<0.01),CoxA 16阳性率分别为62.22%和16.67%; EV71、PE结果差异无统计学意义(P>0.05),实时荧光RT-PCR方法EV71和PE阳性率为42.22%和65.00%,普通RT-PCR方法为37.78%%和55.00%.普通RT-PCR检测的36份EV71、CoxA 16和PE全阴性标本,荧光RT-PCR方法检出PE阳性9份、EV71阳性4份、CoxA 16阳性14份. 结论 实时荧光RT-PCR方法比普通RT-PCR方法更灵敏、快捷,适用于临床手足口病病原体的检测.%Objective To compare and analyze the differences between quantitative RT-PCR (Q-RT-PCR) and conventional RT-PCR testing for hand, foot, and mouth disease (HFMD). Methods Two hundred and fifty-six stool samples were collected from patients clinically diagnosed with HFMD. Viral RNA was extracted and subjected to Q-RT-PCR and conventional RT-PCR for enterovirus 71 (EV71), coxsackievirus A 16 (CoxA 16), and pan-enterovirus (PE) detection. Results were statistically analyzed. Results There was a significant difference in detection of CoxA 16 for Q-RT-PCR and conventional RT-PCR (P0. 05). The rate for Q-RT-PCR detection of EV71 was 42. 22% and that for PE was 65. 00% while the rate for conventional RT-PCR detection of EV71 was 37. 78% and that for PE was 55. 00%. Q-RT-PCR detected 9 PE-positive samples, 4 EV71-positive samples, and 14 CoxA 16-positive samples from a-mong 36 samples that were supposedly negative according conventional RT-PCR. Conclusion Q-RT-PCR was more sensitive and faster than conventional RT-PCR, so Q-RT-PCR is more suitable for detection of

  11. 实时定量RT-PCR对乙型肝炎病毒全长RNA(fRNA)的定量检测%Validation of a Simple Real-time RT-PCR for Detection and Quantization of HBV fRNA

    Institute of Scientific and Technical Information of China (English)

    张佳瑞; 巩丽; 朱少君; 韩秀娟; 姚丽; 王姝妹; 李艳红; 张伟

    2015-01-01

    Objective:To establish a simple assay for the detection and quantization of full-length RNA (fRNA) terminating at polyadenilation site in sera of chronic hepatitis B (CHB) patients.Methods:fRNA were assayed via TaqMan real-time RT/PCR using anchored oligo-dT primers in sera of 53 treatment-naive CHB patients and 22 HBsAg-negative healthy controls.Results were analyzed by comparation ofHBV DNA with HBcrAg and HBeAg.Results:The fRNA assay had a lower limit of detection and quantization at 2.3 log copies/ml,and a correlation coefficient of 0.99 (P<0.0001).fRNA was detected in 29 of 53 (54.7%) of the CHB patients as compared to non of 22 controls (Specificity).fRNA was detected in all 27 HBeAg-positive and/or high HBV DNA levels CHB patients as compared to 2 of 26 (7.7%) HBeAg-negative and low HBV DNA levels CHB patients (P<0.0001).fRNA levels were higher in HBeAg-positive than in HBeAg-negative samples (5.0± 0.3 vs.2.9± 0.4 log copies/ml,P<0.001).Significant correlation was found between fRNA and HBV DNA/HBcrAg (r=0.905 and 0.881,respectively,P<0.0001).The effective items on fRNA levels,in descending order,were:HBV DNA,HBcrAg by means of Hayashi's quantification method type Ⅰ (Multiple correlation efficient=0.939).Conclusion:The simple real-time RT/PCR for detection and quantization of fRNA was suitable for routine clinical test in assessing HBV replication status the same as HBV DNA and HBeAg in CHB patients.%目的:建立一种简便的定量检测慢性乙型肝炎患者血清中终止于聚腺苷酸化位点的乙型肝炎病毒全长RNA(fRNA)的方法.方法:选取53例未治疗的乙型肝炎患者及22例HBsAg阴性的健康者为研究对象,使用锚定oligo-dT的引物对其血清中fRNA进行实时定量反转录PCR检测,统计分析其与HBV DNA、HBcrAg和HBeAg的相关性.结果:对fRNA进行实时荧光定量RT-PCR检测的下线为2.3 log copies/ml,标准曲线的相关系数为0.99(P<0.0001).53例乙型肝炎患者中,29例(54.7

  12. 非洲马瘟病毒VP7和NS2双重荧光RT-PCR检测技术的建立与应用%Development and Application of Duplex Real-time RT-PCR Assay for the Detection of VP7 and NS2 of African Horse Sickness Virus

    Institute of Scientific and Technical Information of China (English)

    高志强; 张鹤晓; 乔彩霞; 蒲静; 张伟; 谷强; 刘环; 张利峰; 马贵平

    2013-01-01

      Nucleic acid sequences of representative strains of different genotypes of African horse sickness viruses(AHSV)were aligned with the DNAMAN software. The two highly conservative NS2 and VP7 regions were then subjected to design primers and probes. The artificially synthesized nucleic acid fragments including amplification regions were used to prepare double strand RNA (dsRNA)by in vitro transcription in two directions(T7 and SP6). A duplex real-time TaqMan RT-PCR assay was developed to detect and quantify AHSV by optimization of reaction conditions using prepared dsRNA. The developed assay was used to detect a set of extracted pathogen RNA/DNA including those of AHSV,Eastern and Western equine encephalomyelitis virus(EEEV and WEEV), equine arteritis virus(EAV),equine influenza virus subtype H3N8(EIV H3N8),Salmonella abortus equi,Streptococcus equi subsp. zooepidimicus resulting in positive only for AHSV RNA,but negative for other virus nucleic acids,suggesting that the developed assay was specific and reliable. The detection limit of the real-time RT-PCR was 1.0×102copies per reaction,10 times more sensitive than that of the conventional RT–PCR based on gel electrophoresis. Also the double gene design could ensure the reliability, and efficiently reduce false negative results. By testing 248 clinical samples,it is confirmed that this assay was rapid,sensitive and repeatable,meeting the requirements for rapid diagnosis of AHSV.%  利用DNAMAN软件对非洲马瘟病毒不同基因型代表株的序列进行分析,选择其高度保守的VP7和NS2基因设计合成引物和探针。人工分别合成包含有扩增区域的VP7和NS2核苷酸片段进行双向(T7和SP6)体外转录制备双链RNA(dsRNA)。使用制备的dsRNA在对荧光定量RT-PCR的反应条件优化的基础上,建立了适用于非洲马瘟病毒检测的双重通用荧光定量RT-PCR检测技术。应用建立的方法对非洲马瘟病毒核酸,马流感病毒核酸

  13. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae.

    Directory of Open Access Journals (Sweden)

    Meng Sun

    Full Text Available The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA, elongation factor 1 (EF1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ribosomal protein S13 (RPS13, ribosomal protein S20 (RPS20, tubulin (TUB, and β-actin (ACTB were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1 were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands. 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults. 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C. To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83 was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  14. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2015-01-01

    The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  15. Enabling Real-Time Analysis at LHCb

    CERN Multimedia

    Govorkova, Katya

    2017-01-01

    A new streaming strategy of the LHCb experiment includes the possibility to perform the physics analysis with candidates reconstructed in the trigger, thus bypassing the offline reconstruction. In the Turbo stream the trigger writes out a compact summary of physics objects containing all information necessary for analyses. This allows an increased output rate and thus higher average efficiencies. The Turbo stream was introduced in 2015 and has allowed for and expanded physics program in Run 2 of the LHC.

  16. Molecular analysis of cell type-specific gene expression profile during mouse spermatogenesis by laser microdissection and qRT-PCR.

    Science.gov (United States)

    Esakky, Prabagaran; Hansen, Deborah A; Drury, Andrea M; Moley, Kelle H

    2013-03-01

    Laser microdissection (LMD) is a selective cell isolation technique that enables the separation of desired homogenous cell subpopulations from complex tissues such as the testes under direct microscopic visualization. The LMD accompanied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) represents an indispensable tool in quantifying messenger RNA (mRNA) expression among defined cell populations. Gene expression is temporally and spatially regulated at 3 sequential phases of mitotic, meiotic, and postmeiotic stages of spermatogenesis. The present study demonstrates a short modified LMD protocol based upon hematoxylin and eosin (H&E) staining. Stage-specific LMD success was validated by the use of mRNA profiling of "marker genes" which are conserved across species and are known to be differentially expressed during spermatogenesis. Magea4, Hspa2, Cox6b2, Tnp1, Prm1, and Prm2 are used to differentiate among the microdissected cell populations, namely spermatogonia (group I), spermatocytes (group II), round and condensing spermatids (group III), and elongated and condensed spermatids (group IV), respectively. The LMD combined with qRT-PCR is further extended to assess the cell stage-specific distribution of selected stress response genes such as Hsp90aa1, Gpx4, Ucp2, Sod1, and Sod2. The germ cell-specific mRNA profiles are suitably complemented by Western blot of the LMD samples, immunohistochemistry, and confocal localization of the corresponding proteins. The current study suggests that LMD can successfully isolate cell subpopulations from the complex tissues of the testes; and establish cell stage-specific basal expression patterns of selected stress response genes and proteins. It is our hypothesis that the baseline expression of stress response genes will differ by cell stage to create discrete stage-specific vulnerabilities to reproductive toxicants.

  17. Analysis of β/α globin ratio by using relative qRT-PCR for diagnosis of beta-thalassemia carriers.

    Science.gov (United States)

    Ranjbaran, Reza; Okhovat, Mohammad Ali; Mobarhanfard, Arash; Aboualizadeh, Farzaneh; Abbasi, Mozhdeh; Moezzi, Leili; Golafshan, Habib Allah; Behzad-Behbahani, Abbas; Bagheri, Mandana; Sharifzadeh, Sedigheh

    2013-07-01

    Current routine tests for premarital screening of β-thalassemia carriers are not applicable for diagnosis of rare atypical minor β-thalassemia cases. A more specialized laboratory evaluation for them is the measurement of β/α chain synthesis ratio with the assistance of radioactive amino acids. This method is also no longer routinely accessible. Consequently it is required to establish a rapid, trouble-free, and reliable method that encompasses all the cases of β-thalassemia carriers. Therefore we have determined β/α-globin mRNA ratio by applying relative qRT-PCR in various β-thalassemia patients. Reticulocytes RNA extraction and subsequent cDNA synthesis were performed, followed by relative qRT-PCR for α- and β-globin chain genes and β-actin gene as an endogenous reference. β/α-Globin gene ratio was then evaluated with the Pfaffl method. The mean of β/α ratio was 0.99, 0.81, 0.69, and 0.69 for normal population, minor, intermediate, and major β-thalassemia, respectively. Approximately 6% of cases with minor thalassemia RBC index and normal HbA2 and having a decreased β/α ratio were located in the minor β-thalassemia group. The mean of β/α mRNA ratio in normal individuals and minor β-thalassemia was significantly different with all other groups (P-value premarital screening test for β-thalassemia carriers. © 2013 Wiley Periodicals, Inc.

  18. Space Shuttle Main Engine real time stability analysis

    Science.gov (United States)

    Kuo, F. Y.

    1993-01-01

    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.

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

    Science.gov (United States)

    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

  20. 西部马脑脊髓炎病毒实时荧光RT-PCR检测方法建立及标准质控品制备%Development of the Real-time RT-PCR for Detection of Western Equine Encephalomyelitis Virus and Preparation of Reference Materials for Quality Control

    Institute of Scientific and Technical Information of China (English)

    谷强; 吴亚琼; 高志强; 刘环; 张伟; 蒲静; 乔彩霞; 张鹤晓; 吴清民

    2013-01-01

    The conserved region was used to design and synthetize primers and TaqMan probe by aligning nucleic acid sequence of representative strains of western equine encephalomyelitis virus (WEEV). A real-time RT-PCR(RRT-PCR) was developed to detect WEEV by the optimization of primers, probe and reaction conditions. The established RRT-PCR was used to detect a panel of extracted virus RNA/DNAs including those of WEEV, eastern equine encephalomyelitis virus(EEEV), equine arteritis virus(EAV), equine herpes virus type 1(EHV-1), equine influenza virus subtype H3N8(EIV H3N8) with only positive result for WEEV RNA, but negative for other four virus DNA/RNAs. Furthermore, cRNA(7442nt~10011nt) of WEEV McMillan strain was prepared by in vitro transcription. After copies calculation, dilution, aliquot, homogeneity and stability testing, the cRNAs were used to evaluate the established RRT-PCR. The result indicated that the detection limit of RRT-PCR was 10 copies per reaction using in vitro transcribed cRNA, It only took 4h to detect 197 blood samples from imported horses, suggesting that the RRT-PCR was a rapid, sensitive and specific method, and the method can be used as a technical reserve for WEEV screening of imported horses.%选取西部马脑炎病毒代表株进行序列比对分析,选择保守区域,设计合成引物和TaqMan探针。经对反应体系和条件进行优化,建立了检测西部马脑炎病毒的实时荧光RT-PCR检测方法,应用建立的方法对西部马脑炎病毒核酸、东部马脑炎病毒核酸、马动脉炎病毒核酸、马疱疹病毒1型核酸、马流感病毒H3N8亚型核酸进行检测,结果表明建立的方法只能检出西部马脑炎病毒核酸,与另外4种病毒核酸无交叉反应。进一步通过体外转录制备了西部马脑炎病毒McMillan株7442~10011的cRNA片段,经拷贝数计算、稀释、分装、均匀性和稳定性检验后,作为质控品对建立的方法进行评价。结果显示,所

  1. Analysis and Synthesis of Distributed Real-Time Embedded Systems

    DEFF Research Database (Denmark)

    Pop, Paul; Eles, Petru; Peng, Zebo

    Embedded computer systems are now everywhere: from alarm clocks to PDAs, from mobile phones to cars, almost all the devices we use are controlled by embedded computers. An important class of embedded computer systems is that of hard real-time systems, which have to fulfill strict timing...... in important reductions of design costs. Analysis and Synthesis of Distributed Real-Time Embedded Systems will be of interest to advanced undergraduates, graduate students, researchers and designers involved in the field of embedded systems....

  2. Development of novel AllGlo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9.

    Science.gov (United States)

    Zhang, Yanjun; Mao, Haiyan; Yan, Juying; Wang, Xinying; Zhang, Lei; Guus, Koch; Li, Hui; Li, Zhen; Chen, Yin; Gong, Liming; Chen, Zhiping; Xia, Shichang

    2014-07-01

    Recently, human deaths have resulted from infection with low-pathogenicity avian influenza virus H7N9 strains that have emerged recently in China. To strengthen H7N9 surveillance and outbreak control, rapid and reliable diagnostic methods are needed. To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of H7N9 viral RNA, primers and AllGlo probes were designed to target the HA and NA genes of H7N9. Conserved sequences in the HA and NA genes were identified by phylogenic analysis and used as targets for H7N9 virus detection. The similarities of the targeted HA and NA gene sequences from different H7 and N9 influenza virus strains were 93.2-99.9 % and 96.0-99.6 %, respectively The specificity and sensitivity of the new multiplex real-time qRT-PCR was established. The test was used for the detection of viral RNA in human pharyngeal swabs and environmental samples. The detection limit of the multiplex qRT-PCR was estimated to be about 10(-1) TCID50/reaction. Finally, the diagnostic sensitivities of the multiplex qRT-PCR, virus isolation and TaqMan qRT-PCR were compared using pharyngeal swabs and environmental samples. These analyses yielded positive results in 46.7 %, 43.3 % and 20.0 % of the samples, respectively. The novel multiplex AllGlo qRT-PCR is a rapid and sensitive method to identify H7N9 virus in clinical and environmental samples and can be used to facilitate studies on the epidemiology of H7N9 virus.

  3. Development of automated system for real-time LIBS analysis

    Science.gov (United States)

    Mazalan, Elham; Ali, Jalil; Tufail, Kashif; Haider, Zuhaib

    2017-03-01

    Recent developments in Laser Induced Breakdown Spectroscopy (LIBS) instrumentation allow the acquisition of several spectra in a second. The dataset from a typical LIBS experiment can consist of a few thousands of spectra. To extract the useful information from that dataset is painstaking effort and time consuming process. Most of the currently available softwares for spectral data analysis are expensive and used for offline data analysis. LabVIEW software compatible with spectrometer (in this case Ocean Optics Maya pro spectrometer), can be used to for data acquisition and real time analysis. In the present work, a LabVIEW based automated system for real-time LIBS analysis integrated with spectrometer device is developed. This system is capable of performing real time analysis based on as-acquired LIBS spectra. Here, we have demonstrated the LIBS data acquisition and real time calculations of plasma temperature and electron density. Data plots and variations in spectral intensity in response to laser energy were observed on LabVIEW monitor interface. Routine laboratory samples of brass and calcine bone were utilized in this experiment. Developed program has shown impressive performance in real time data acquisition and analysis.

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

    Science.gov (United States)

    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. Copyright © 2016. Published by Elsevier Masson SAS.

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

    Science.gov (United States)

    Wang, Huili; Yang, Yanmei; Chen, Wei; Ding, Li; Li, Peizhen; Zhao, Xiaokai; Wang, Xuedong; Li, Aiying; Bao, Qiyu

    2013-01-30

    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.

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

    Science.gov (United States)

    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

  7. [Microarray analytic system for multiplex analysis by real-time polymerase chain reaction with reagents immobilized in microreactors].

    Science.gov (United States)

    Navolotskiĭ, D V; Perchik, A V; Mark'ianov, I A; Ganeev, A A; Sliadnev, M N

    2011-01-01

    A microarray analytic system that uses a silicon chip with immobilized in microreactor test-system for multiplex analysis of DNA by real-time polymerase chain reaction (RT-PCR) was developed and optimized. We suggested the method of immobilization of PCR-components of a test-system, chose the stabilizer, and conducted the optimization of the composition of reaction mixture to achieve permanent stability of a microarray. We conducted optimization of preparation of samples using magnetic sorbent and indicated that, with 2.6 x 10(4) copies/ml, 60 min are necessary to obtain positive identification including time for preparation of model probes. The abilities of the created system were demonstrated on the example of microarray analysis of samples with different content of DNA, low absolute limits of identification (20 DNA copies in microreactor), and high reproducibility of the analysis.

  8. Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2016-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq® DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix. PMID:22975959

  9. DGGE and real-time PCR analysis of lactic acid bacteria in bacterial communities of the phyllosphere of lettuce.

    Science.gov (United States)

    Zwielehner, Jutta; Handschur, Michael; Michaelsen, Astrid; Irez, Selen; Demel, Michael; Denner, Ewald B M; Haslberger, Alexander G

    2008-05-01

    Food associated indigenous microbial communities exert antagonistic effects on pathogens and may routinely deliver health relevant microorganisms to the GI tract. By using molecular, culture independent methods including PCR-DGGE of 16S rDNA-coding regions and real-time PCR (RT-PCR) as well as BIOLOG metabolic fingerprinting, microbial communities on lettuce were analyzed in samples from fields, from supermarkets and soil. Amplified 16S rRNA gene sequences (57.7%) could be assigned to species previously reported as typical for the phyllosphere including Pantoea agglomerans, Pseudomonas flavescens, Moraxella spp., and Mycobacterium spp. 71.8% of the sequences obtained represented so far undescribed taxa. Principal component analysis of BIOLOG metabolic profiles indicated a seasonal variation in the lettuce phyllosphere microbial community structure. Various lactic acid bacteria were detected including several Lactobacillus and Leuconostoc species in particular on lettuce from organic farming. By RT-PCR lactobacilli were found with a range of abundances from 1x10(4 )to 1x10(5 )copies/g lettuce. Considering the importance of salad in many diets lettuce may contribute to a constant supply with LAB.

  10. Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score

    Science.gov (United States)

    Tekin, Nilgun; Conget, Paulette; Bruna, Flavia; Timar, Botond; Gagyi, Eva; Basak, Ranjan; Naik, Omkar; Auewarakul, Chirayu; Sritana, Narongrit; Levy, Debora; Cerci, Juliano Julio; Bydlowski, Sergio Paulo; Pereira, Juliana; Dimamay, Mark Pierre; Natividad, Filipinas; Chung, June-Key; Belder, Nevin; Kuzu, Isinsu; Paez, Diana; Dondi, Maurizio; Carr, Robert

    2016-01-01

    As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting. PMID:27825111

  11. Comparison of the Roche RealTime ready Influenza A/H1N1 Detection Set with CDC A/H1N1pdm09 RT-PCR on samples from three hospitals in Ho Chi Minh City, Vietnam

    Science.gov (United States)

    Tham, Nguyen thi; Hang, Vu thi Ty; Khanh, Trong Huu; Viet, Do Chau; Hien, Tran Tinh; Farrar, Jeremy; van Vinh Chau, Nguyen; van Doorn, H. Rogier

    2012-01-01

    Background Real-time PCR can be considered the gold standard for detection of influenza viruses due to its high sensitivity and specificity. Roche has developed the RealTime ready Influenza A/H1N1 Detection Set, consisting of a generic influenza virus A PCR targeting the M2 gene (M2 PCR) and a specific PCR targeting the HA of A/H1N1-pdm09 (HA PCR, 2009 H1N1), with the intention to make a reliable, rapid, and simple test to detect and quantify 2009 H1N1 in clinical samples. Methods We evaluated this kit against the USCDC/WHO real-time PCR for influenza virus using 419 nose and throat swabs from 210 patients collected in 3 large hospitals in Ho Chi Minh city, Vietnam. Results In the per patient analysis, when compared to CDC PCR, the sensitivity and specificity of the M2 PCR were 85.8 and 97.6%, respectively; the sensitivity and specificity of HA PCR were 88.2 and 100%, respectively. In the per sample analysis, the sensitivity and specificity in nose swabs were higher than in throat swabs for both M2 and HA PCRs. The viral loads as determined with the M2 and HA PCRs correlated well with the Ct values of the CDC PCR. Conclusion Compared with the CDC PCR, the kit has a reasonable sensitivity and very good specificity for the detection and quantification of Influenza A virus and A/H1N1-pdm09. However, given the current status of 2009 H1N1, a kit that can detect all circulating seasonal influenza viruses would be preferable. PMID:22785431

  12. RT-PCR Analysis of ED-A,ED-B, and IIICS Fibronectin Domains: A New Screening Marker For Bladder Cancer

    Directory of Open Access Journals (Sweden)

    M Ahmadi Javid

    2004-12-01

    Full Text Available Background: Fibronectin seems to play a very important role in the progression and invasion of bladder cancer. EDA, EDB, and IIICS domains of fibronectin are not expressed in the adult persons but they’re expressed in different cancers. The aim of this study is to investigate the mRNA of fibronectin in transitional carcinoma cells (TCC of bladder to study these domains. Methods: A total of 20 patients with known bladder cancer were studied. Two of them excluded since their excised tissues were not enough for both the pathological examination and RNA study. Another 20 (control group were normal volunteers who needed bladder operations. The excised tissue was immediately transferred to RNAlater (Ambion,TX. RNA was extracted via RNAWIZ (Ambion, TX. cDNA was made via RevertAid First Strand cDNA Synthesis Kit (Fermentas. PCR of the cDNAs was performed using primers for EDA, EDB, and IIICS (Eurogentec,Belgium. Results: For the first time, we present the expression of the oncofetal fibronectin mRNA in the transitional cell carcinoma of bladder. The high grade muscle invasive (G3T2 tumor, expressed ED-A, ED-B, and IIICS. Expression of ED-A, ED-B, and IIICS was confirmed in the two patients with G3T1 TCC. The four patients with G2Ta and G3Ta expressed both ED-A and ED-B. The four patients with G1T1 tumor expressed ED-A only, similar to the nine patients with G1Ta tumor. None of the normal volunteers expressed the oncofetal extra domains. The sensitivity of ED-A positive fibronectin RNA for detecting TCC of any kind is 100%, and of ED-B was only 35%. The specificity of ED-B positive fibronectin RNA for the high grade TCC is 100%. Conclusion: ED-A, ED-B, and IIICS could be used as useful markers for the diagnosis and following up of bladder carcinoma. Keywords: Transitional Cell Carcinoma, bladder cancer, fibronectin, RT-PCR, oncofetal.

  13. Modelling and analysis of real-time and hybrid systems

    Energy Technology Data Exchange (ETDEWEB)

    Olivero, A.

    1994-09-29

    This work deals with the modelling and analysis of real-time and hybrid systems. We first present the timed-graphs as model for the real-time systems and we recall the basic notions of the analysis of real-time systems. We describe the temporal properties on the timed-graphs using TCTL formulas. We consider two methods for property verification: in one hand we study the symbolic model-checking (based on backward analysis) and in the other hand we propose a verification method derived of the construction of the simulation graph (based on forward analysis). Both methods have been implemented within the KRONOS verification tool. Their application for the automatic verification on several real-time systems confirms the practical interest of our approach. In a second part we study the hybrid systems, systems combining discrete components with continuous ones. As in the general case the analysis of this king of systems is not decidable, we identify two sub-classes of hybrid systems and we give a construction based method for the generation of a timed-graph from an element into the sub-classes. We prove that in one case the timed-graph obtained is bi-similar with the considered system and that there exists a simulation in the other case. These relationships allow the application of the described technics on the hybrid systems into the defined sub-classes. (authors). 60 refs., 43 figs., 8 tabs., 2 annexes.

  14. 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).

    Science.gov (United States)

    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.

  15. Salmonella detection from chicken rinsate with surface enhanced Raman spectroscopy and RT-PCR validation

    Science.gov (United States)

    Optical detection of bacteria has been approached in recent years as a bacteria detection method that can counter time restraints of traditional plating or the high reoccurring cost of real-time polymerase chain reaction (RT-PCR). The goal of optical detection is to identify bacteria with spectral s...

  16. Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba skin biopsies

    Directory of Open Access Journals (Sweden)

    Casini Silvia

    2006-09-01

    Full Text Available Abstract Background Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. Results Ten commonly used housekeeping genes (HKGs were partially sequenced in the striped dolphin (Stenella coeruleoalba and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH and tyrosine 3-monooxygenase (YWHAZ always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4 and S18 (RPS18 also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC, phosphoglycerate kinase 1 (PGK1, hypoxanthine ribosyltransferase (HPRT1 and β-2-microglobin (B2M show variable expression

  17. Interactive Construction Digital Tools With Real Time Analysis

    DEFF Research Database (Denmark)

    Klitgaard, Jens; Kirkegaard, Poul Henning

    2007-01-01

    . An example of a prototype for a digital conceptual design tool with integrated real time structural analysis is presented and compared with a more common Building Information Modelling (BIM) approach. It is concluded that a digital conceptual design tool with embedded real time structural analysis could......The recent developments in computational design tools have evolved into a sometimes purely digital process which opens up for new perspectives and problems in the sketching process. One of the interesting possibilities lay within the hybrid practitioner- or architect-engineer approach, where...... an architect-engineer or hybrid practitioner works simultaneously with both aesthetic and technical design requirements. In this paper the problem of a vague or not existing link between digital design tools, used by architects and designers, and the analysis tools developed by and for engineers is considered...

  18. Analysis and Optimization of Heterogeneous Real-Time Embedded Systems

    DEFF Research Database (Denmark)

    Pop, Paul; Eles, Petru; Peng, Zebo

    2005-01-01

    . The success of such new design methods depends on the availability of analysis and optimization techniques. In this paper, we present analysis and optimization techniques for heterogeneous real-time embedded systems. We address in more detail a particular class of such systems called multi-clusters, composed......An increasing number of real-time applications are today implemented using distributed heterogeneous architectures composed of interconnected networks of processors. The systems are heterogeneous not only in terms of hardware components, but also in terms of communication protocols and scheduling...... of several networks interconnected via gateways. We present a schedulability analysis for safety-critical applications distributed on multi-cluster systems and briefly highlight characteristic design optimization problems: the partitioning and mapping of functionality, and the packing of application messages...

  19. Analysis and optimisation of heterogeneous real-time embedded systems

    DEFF Research Database (Denmark)

    Pop, Paul; Eles, Petru; Peng, Zebo

    2005-01-01

    . The success of such new design methods depends on the availability of analysis and optimisation techniques. Analysis and optimisation techniques for heterogeneous real-time embedded systems are presented in the paper. The authors address in more detail a particular class of such systems called multi......An increasing number of real-time applications are today implemented using distributed heterogeneous architectures composed of interconnected networks of processors. The systems are heterogeneous, not only in terms of hardware components, but also in terms of communication protocols and scheduling......-clusters, composed of several networks interconnected via gateways. They present a schedulability analysis for safety-critical applications distributed on multi-cluster systems and briefly highlight characteristic design optimisation problems: the partitioning and mapping of functionality, and the packing...

  20. Analysis and optimisation of heterogeneous real-time embedded systems

    DEFF Research Database (Denmark)

    Pop, Paul; Eles, Petru; Peng, Zebo

    2006-01-01

    . The success of such new design methods depends on the availability of analysis and optimisation techniques. Analysis and optimisation techniques for heterogeneous real-time embedded systems are presented in the paper. The authors address in more detail a particular class of such systems called multi......An increasing number of real-time applications are today implemented using distributed heterogeneous architectures composed of interconnected networks of processors. The systems are heterogeneous, not only in terms of hardware components, but also in terms of communication protocols and scheduling......-clusters, composed of several networks interconnected via gateways. They present a schedulability analysis for safety-critical applications distributed on multi-cluster systems and briefly highlight characteristic design optimisation problems: the partitioning and mapping of functionality, and the packing...

  1. Real Time Engineering Analysis Based on a Generative Component Implementation

    DEFF Research Database (Denmark)

    Kirkegaard, Poul Henning; Klitgaard, Jens

    2007-01-01

    The present paper outlines the idea of a conceptual design tool with real time engineering analysis which can be used in the early conceptual design phase. The tool is based on a parametric approach using Generative Components with embedded structural analysis. Each of these components uses...... without jumping from aesthetics to structural digital design tools and back, but to work with both simultaneously and real time. The engineering level of knowledge is incorporated at a conceptual thinking level, i.e. qualitative information is used in stead of using quantitative information. An example...... with a static determinate roof structure modelled by beam components is given. The example outlines the idea of the tool for conceptual design in early phase of a multidisciplinary design process between architecture and structural engineering....

  2. Quantitative real-time single particle analysis of virions.

    Science.gov (United States)

    Heider, Susanne; Metzner, Christoph

    2014-08-01

    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.

  3. Real-time analysis of water movement in plant sample

    Energy Technology Data Exchange (ETDEWEB)

    Yokota, Harumi; Furukawa, Jun; Tanoi, Keitaro [Graduate School, Tokyo Univ. (Japan)

    2000-07-01

    To know the effect of drought stress on two cultivars of cowpea, drought tolerant (DT) and drought sensitive (DS), and to estimate vanadium treatment on plant activity, we performed real time{sup 18}F labeled water uptake measurement by PETIS. Fluoride-18 was produced by bombarding a cubic ice target with 50 MeV protons using TIARA AVF cyclotron. Then {sup 18}F labeled water was applied to investigate water movement in a cowpea plant. Real time water uptake manner could be monitored by PETIS. After the analysis by PETIS, we also measured the distribution of {sup 18}F in a whole plant by BAS. When a cowpea plant was treated with drought stress, there was a difference in water uptake manner between DT and DS cultivar. When a cowpea plant was treated with V for 20 hours before the water uptake experiment, the total amount of {sup 18}F labeled water absorption was found to be drastically decreased. (author)

  4. Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions

    OpenAIRE

    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 un...

  5. 苦瓜植株不同组织中降糖血糖多肽活性成分的RT-PCR分析%RT-PCR Analysis of Hypoglycemic Polypeptide Components from Different Tissues of Momordica charantia

    Institute of Scientific and Technical Information of China (English)

    宋还雷; 谢先文; 顾红艳; 任大云; 王富军; 赵健

    2011-01-01

    Objective To investigate the distribution of hypoglycemic polypeptide P in different tissues of Momordica chamntia (MC). Methods The improved one-step Trizol extraction method was used for the isolation of total RNA from different tissues of MC. A RT-PCR method was used for analyzing the expression patterns of the hypoglycemic polypeptide P gene in MC. Results The RT-PCR analysis results showed that the expression of hypoglycemic polypeptide P gene could only be detected in seeds and fruit pulp of mature MC. Conclusion The hypoglycemic polypeptide P mainly expresses in seeds and fruit pulp of MC. These two tissues are the main medicinal parts for hypoglycemic therapy of MC.%目的 研究苦瓜降糖活性多肽P在苦瓜不同组织器官中的分布.方法 用改良Trizol一步法提取苦瓜不同组织的总RNA,采用RT-PCR方法扩增降糖多肽P基因,分析其表达.结果 成熟果实的种子和果肉中均可明显检测到降糖多肽P基因的表达,其他组织中则未能检测出.结论 苦瓜降糖多肽P活性成分主要在苦瓜果肉和种子中表达,这2种组织是苦瓜降血糖作用的主要药用部位.

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

    Science.gov (United States)

    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.

  7. TaqMan荧光定量RT-PCR检测猪脂蛋白酯酶mRNA方法的建立%Establishment of Real-time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

    Institute of Scientific and Technical Information of China (English)

    廉红霞; 卢德勋; 高民

    2008-01-01

    [目的]克隆猪cDNA,作为猪LPL mRNA定量检测的标准品,建立检测方法.[方法]用RT-PCR,从猪背最长肌的总RNA中逆转录扩增LPL的cDNA,将纯化的LPL cDNA与pGM-T载体进行连接,转化宿主菌TOP10,提取重组质粒DNA,PCR鉴定并测序分析,对质粒标准进行实时荧光定量PCR检测.纯化质粒并检测260 nm吸光度,确定原液的重组质粒拷贝浓度并以此制备荧光定量PCR梯度浓度标准品.[结果]建立了猪LPL mRNA实时定量PCR检测方法,特异性好,检测灵敏度达103拷贝,线性范围为1×103~1×1010拷贝,阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系(R2=0.9871).[结论]成功克隆了LPL实时荧光PCR定量标准,且TaqMan荧光定量RT-PCR的方法可对猪背最长肌LPL mRNA的表达进行准确定量.

  8. Self-Timed Scheduling Analysis for Real-Time Applications

    Directory of Open Access Journals (Sweden)

    Marco J. G. Bekooij

    2007-01-01

    Full Text Available This paper deals with the scheduling analysis of hard real-time streaming applications. These applications are mapped onto a heterogeneous multiprocessor system-on-chip (MPSoC, where we must jointly meet the timing requirements of several jobs. Each job is independently activated and processes streams at its own rate. The dynamic starting and stopping of jobs necessitates the usage of self-timed schedules (STSs. By modeling job implementations using multirate data flow (MRDF graph semantics, real-time analysis can be performed. Traditionally, temporal analysis of STSs for MRDF graphs only aims at evaluating the average throughput. It does not cope well with latency, and it does not take into account the temporal behavior during the initial transient phase. In this paper, we establish an important property of STSs: the initiation times of actors in an STS are bounded by the initiation times of the same actors in any static periodic schedule of the same job; based on this property, we show how to guarantee strictly periodic behavior of a task within a self-timed implementation; then, we provide useful bounds on maximum latency for jobs with periodic, sporadic, and bursty sources, as well as a technique to check latency requirements. We present two case studies that exemplify the application of these techniques: a simplified channel equalizer and a wireless LAN receiver.

  9. 甲型H1N1流感病毒两种方法检测限的比较%The detection limit research of H1N1 influenza virus by RT-PCR and real-time pcr

    Institute of Scientific and Technical Information of China (English)

    赵英杰; 张继军; 刘萍; 施文静

    2010-01-01

    目的:比较RT-PCR和Real time PCR检测甲型H1N1流感病毒的灵敏度.方法:体外转录生成甲型H1N1流感病毒M基因235 bp的保守序列作为阳性毒株RNA为模板,计算出相应的拷贝数后进行10倍梯度稀释,在引物探针的作用下分别进行RT-PCR和Real time PCR的检测.结果:Real time PCR的最低检测限为8 copies/μl,RT-PCR的最低检测限为80 copies/μl,线性关系好.结论:检测甲型H1N1流感病毒Real time PCR方法比RT-PCR灵敏度高10倍.

  10. Real time video analysis to monitor neonatal medical condition

    Science.gov (United States)

    Shirvaikar, Mukul; Paydarfar, David; Indic, Premananda

    2017-05-01

    One in eight live births in the United States is premature and these infants have complications leading to life threatening events such as apnea (pauses in breathing), bradycardia (slowness of heart) and hypoxia (oxygen desaturation). Infant movement pattern has been hypothesized as an important predictive marker for these life threatening events. Thus estimation of movement along with behavioral states, as a precursor of life threatening events, can be useful for risk stratification of infants as well as for effective management of disease state. However, more important and challenging is the determination of the behavioral state of the infant. This information includes important cues such as sleep position and the status of the eyes, which are important markers for neonatal neurodevelopment state. This paper explores the feasibility of using real time video analysis to monitor the condition of premature infants. The image of the infant can be segmented into regions to localize and focus on specific areas of interest. Analysis of the segmented regions can be performed to identify different parts of the body including the face, arms, legs and torso. This is necessary due to real-time processing speed considerations. Such a monitoring system would be of great benefit as an aide to medical staff in neonatal hospital settings requiring constant surveillance. Any such system would have to satisfy extremely stringent reliability and accuracy requirements, before it can be deployed in a hospital care unit, due to obvious reasons. The effect of lighting conditions and interference will have to be mitigated to achieve such performance.

  11. Restriction enzyme analysis of RT-PCR amplicons as a rapid method for detection of genetic diversity among bovine respiratory syncytial virus isolates

    NARCIS (Netherlands)

    Valentova, V.; Antonis, A.F.G.; Kovarcik, K.

    2005-01-01

    Our current knowledge of antigenic variability of the bovine respiratory syncytial virus (BRSV) is quite limited and is mainly dependent on the use of monoclonal antibodies (mAb). In this study, we present not only analysis of the antigenic, but also of the genetic variability of BRSV. Using a panel

  12. THE IMPROVEMENT OF RT-PCR TECHNIQUE ON DETECTING ROTAVIRUS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To establish a speed and effective method to detect rotavirus. Methods Using ELISA and one step RT-PCR to detect 196 clinic samples from Xi'an area. Results Compared with ELISA method, one step RT PCR was more sensitive and specific (P <0.05). Conclusion One step RT-PCR is a simple, speed, sensitive and spe cific method for clinic and epidemic studies of rotavirus.

  13. Implementation and Analysis of Real-Time Streaming Protocols.

    Science.gov (United States)

    Santos-González, Iván; Rivero-García, Alexandra; Molina-Gil, Jezabel; Caballero-Gil, Pino

    2017-04-12

    Communication media have become the primary way of interaction thanks to the discovery and innovation of many new technologies. One of the most widely used communication systems today is video streaming, which is constantly evolving. Such communications are a good alternative to face-to-face meetings, and are therefore very useful for coping with many problems caused by distance. However, they suffer from different issues such as bandwidth limitation, network congestion, energy efficiency, cost, reliability and connectivity. Hence, the quality of service and the quality of experience are considered the two most important issues for this type of communication. This work presents a complete comparative study of two of the most used protocols of video streaming, Real Time Streaming Protocol (RTSP) and the Web Real-Time Communication (WebRTC). In addition, this paper proposes two new mobile applications that implement those protocols in Android whose objective is to know how they are influenced by the aspects that most affect the streaming quality of service, which are the connection establishment time and the stream reception time. The new video streaming applications are also compared with the most popular video streaming applications for Android, and the experimental results of the analysis show that the developed WebRTC implementation improves the performance of the most popular video streaming applications with respect to the stream packet delay.

  14. Resource-Parameterized Timing Analysis of Real-Time Systems

    DEFF Research Database (Denmark)

    Kim, Jin Hyun; Legay, Axel; Larsen, Kim Guldstrand

    2015-01-01

    Cyber-Physical Systems (CPS) are subject to platform-given resource constraints upon such resources as CPU, memory, and bus, in executing their functionalities. This causes the behavior of a verified application to deviate from its intended timing behavior when the application is integrated...... on a specic platform. For the same reason, a configuration of platforms cannot be independent from applications in most cases. This paper proposes a new analysis framework of real-time systems where an application and a platform can be analyzed in a fully independent way such that not only the application...... be parameterized by various resource congurations. For analysis of application and platform models, we use two model checking techniques: symbolic and statistical model checking techniques of Uppaal. Our framework is demonstrated by a case study where a turn indicator system is analyzed with respect to various...

  15. Real Time Engineering Analysis Based on a Generative Component Implementation

    DEFF Research Database (Denmark)

    Kirkegaard, Poul Henning; Klitgaard, Jens

    2007-01-01

    The present paper outlines the idea of a conceptual design tool with real time engineering analysis which can be used in the early conceptual design phase. The tool is based on a parametric approach using Generative Components with embedded structural analysis. Each of these components uses...... the geometry, material properties and fixed point characteristics to calculate the dimensions and subsequent feasibility of any architectural design. The proposed conceptual design tool provides the possibility for the architect to work with both the aesthetic as well as the structural aspects of architecture...... with a static determinate roof structure modelled by beam components is given. The example outlines the idea of the tool for conceptual design in early phase of a multidisciplinary design process between architecture and structural engineering....

  16. Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

    Science.gov (United States)

    Shi, Yan; Li, Li-Zhen; Sun, Jian-Zhi; Zhang, Ti; Peng, Jun; Xu, Cong-Gao

    2008-01-01

    Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

  17. Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR

    DEFF Research Database (Denmark)

    King, Donald P.; Montague, Nick; Ebert, Katja

    2007-01-01

    This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot...... unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs...... did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants...

  18. Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR

    DEFF Research Database (Denmark)

    King, Donald P.; Montague, Nick; Ebert, Katja

    2007-01-01

    did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants......This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot...... unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs...

  19. Space Shuttle telemetry analysis by a real time expert system

    Science.gov (United States)

    Muratore, John F.

    1987-01-01

    During early manned spacecraft operations, the primary role of ground telemetry systems was data display to flight controllers. As manned spaceflights have increased in complexity, greater demands have been placed on flight controllers to simultaneously monitor systems and replan systems operations. This has led to interest in automated telemetry monitoring systems to decrease the workload on flight controllers. The Mission Operations Directorate at the Lyndon B. Johnson Space Center has developed a five layer model to integrate various monitoring and analysis technologies such as digital filtering, fault detection algorithms, and expert systems. The paper describes the five layer model and explains how it has been used to guide prototyping efforts at Mission Control. Results from some initial expert systems are presented. The paper also describes the integrated prototype currently under development which implements a real time expert system to assist flight controllers in the Mission Control Center in monitoring Space Shuttle communications systems.

  20. Method of Real-Time Principal-Component Analysis

    Science.gov (United States)

    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.

  1. VLBI real-time analysis by Kalman Filtering

    Science.gov (United States)

    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.

  2. 鸭肝炎病毒GD株的分离与RT-PCR鉴定分析%Isolation and Identification Analysis of Duck Hepatitis Virus GD Strain by RT-PCR

    Institute of Scientific and Technical Information of China (English)

    黄昌力; 魏春娅; 张超轶; 袁镜乐; 刘志彬; 张桂红

    2012-01-01

    In order to complete a duck hepatitis virus (DHV) stain's isolation and sero-genotyping research, the suspected DHV liver from died duckling in Guangdong province was selected as sample, and then was carried on regression analysis, ELD50 determination and duck embryo neutralization test after viral isolation, duck embryo inoculation and purification. The results showed that the serotype of the duck hepatitis virus was type Ⅰ. According to RT-PCR detect and sequence analysis, it was also identified as genetype Ⅰ of DHV. Named the isolated virus strain as GD.%本研究分离了1株鸭肝炎病毒(duck hepatitis virus,DHV),并对其进行了分型研究.取广东某鸭场疑似DHV致死8日龄雏鸭肝脏,肝脏组织处理后经鸭胚接种分离纯化病毒.分离毒株经动物回归试验、ELD5o测定、中和试验等确定为血清Ⅰ型DHV,同时通过RT-PCR检测及序列分析,从基因分型角度也验证分离毒株为DHV-Ⅰ,并将该分离毒命名为GD株.

  3. Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

    Directory of Open Access Journals (Sweden)

    Pengfei Lin

    Full Text Available The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

  4. Relative Quantification of mRNA Transcription of αv Subunits Relevant to Food and mouth disease virus (FMDV) Receptors in Different Tissues of Sheep by Real-time Quantitative RT-PCR%绵羊口蹄疫病毒整联蛋白受体αv亚基基因不同组织表达差异

    Institute of Scientific and Technical Information of China (English)

    吾热力哈孜; 陈创夫; 徐宁迎; 胡圣伟; 王遵宝; 马柿委

    2011-01-01

    为了建立检测绵羊口蹄疫病毒(Foot andmouth disease virus,FMDV)整联蛋白受体αv亚基基因mRNA相对表达量的荧光定量RT-PCR方法,本研究根据报道的绵羊FMDV整联蛋白受体αv亚基(integrin,alphaV,ITGAV)基因序列设计实时定量PCR引物,以β-αctin为内参基因,采用相对荧光定量RT-PCR方法,检测分析αv基因在绵羊体内不同组织器官中的mRNA表达谱.结果显示αv基因在绵羊19种组织中均有不同程度的转录表达,在乳腺组织中表达量最高,蹄组织次之,肌肉组织最少,卵巢、瘤胃、气管、小肠、肺等组织上也有不同程度的表达.本研究成功建立了检测FMDV整联蛋白受体αv亚基基因在绵羊不同器官组织中mRNA表达水平的相对荧光定量RT-PCR方法,并明确了αv基因在不同组织间的表达差异,为FMDV组织嗜性研究及分子生物学检测方法的建立提供了资料.%For establishing an assay of Real-time PCR to detect distribution and expression level of sheep Food and mouth disease virus (MDV) integrin receptor αv submit in different tissue, in this study, a pair of primers was designed according to the published nudeotide sequence of integrin receptor αv submit.And tissue expression profile of sheep (Ovisaries) integrin αv in different tissue was detected by Real-time PCR.The results showed that the integrin receptor αv submit was generally expressed in 19 kinds of tissues with different level.The highest expression level was showed in breast, the next was in hoof, and the lowest was in muscle tissue.We have established the Real-time PCR method which can be used to confirm tissue profile of sheep FMDV integrin receptor αv submit.This study provides basic data for further study tissue tropism of FMDV and molecular detection method.

  5. Microarray and quantitative RT-PCR analyses in calcium-channel blockers induced gingival overgrowth tissues of periodontitis patients.

    Science.gov (United States)

    Shimizu, Taro; Kubota, Takehiko; Nakasone, Naohiro; Abe, Daisuke; Morozumi, Toshiya; Yoshie, Hiromasa

    2011-03-01

    The purpose of the present study was to analyse transcriptomes and mRNA expression levels for specific genes in calcium-channel blocker-induced gingival overgrowth (GO) tissues. Eight gingival tissues samples (from both GO negative and positive sites) were harvested from four GO patients for microarray analyses. Twelve candidate genes were selected for further quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) analyses. Ten GO tissues from periodontitis patients and ten control gingival tissues from healthy subjects were compared by qRT-PCR. Mann-Whitney U-test was used for statistical evaluation. In GO positive tissues, 163-1631 up-regulated and 100-695 down-regulated genes were identified with more than two-fold changes compared with GO negative tissues amongst patients by microarray experiments. No commonly expressed genes amongst the eight sets of microarray data were found. The clustering analysis confirmed that the entire transcriptome patterns showed similarities in individuals, but differences amongst the four patients. The qRT-PCR and statistical analyses for the candidate genes, though, revealed differential gene expressions between GO-positive and negative tissues. We found that matrix metalloproteinase (MMP)-1 and MMP-12 as well as cathepsin-L were significantly up-regulated whilst keratin-10 and transforming growth factor-β1 were significantly down-regulated in GO tissues of periodontitis patients compared with the control gingival tissues of healthy subjects. The microarray analyses revealed that GO pathogenesis was complex and individually varied, though GO-affected gingival tissues were controlled at least by genes related to collagen metabolisms including regulated MMPs, cathepsin-L, growth factors, and keratins to maintain tissue homeostasis in vivo. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Cluster Computing For Real Time Seismic Array Analysis.

    Science.gov (United States)

    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

  7. 实时荧光定量PCR检测端粒酶逆转录酶mRNA在急性白血病中的表达%Expression of Telomerase Reverse Transcriptase mRNA in Acute Leukemia and Its Significance Detected by Quantitative Real-time RT-PCR

    Institute of Scientific and Technical Information of China (English)

    汤立旦; 黄河; 孙洁

    2006-01-01

    目的研究人类端粒酶转录酶(hTERT)在急性白血病的表达和意义.方法应用实时定量PCR(Real-time PCR)技术检测30例急性白血病骨髓单个核细胞及25例正常人骨髓单个核细胞中hTERT mRNA的表达水平,分析其表达水平与急性白血病恶性程度的关系.结果 hTERT在急性白血病细胞中的中位表达量为0.00244(0~0.0320),明显高于正常人骨髓单个核细胞中hTERT的中位表达量2.79×10-4(0~0.0078)(P<0.001).但在ANLL和ALL细胞中对比表达无明显差异(P=0.742).结论在急性白血病中hTERT明显高于正常对照,hTERT是急性白血病诊断的特异性指标之一.

  8. Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.

    Science.gov (United States)

    Castiglione, Francesca; Rossi Degl'Innocenti, Duccio; Taddei, Antonio; Garbini, Francesca; Buccoliero, Anna Maria; Raspollini, Maria Rosaria; Pepi, Monica; Paglierani, Milena; Asirelli, Grazia; Freschi, Giancarlo; Bechi, Paolo; Taddei, Gian Luigi

    2007-09-01

    In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.

  9. Predicting Gene Structures from Multiple RT-PCR Tests

    Science.gov (United States)

    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.

  10. Analysis of hybrid viscous damper by real time hybrid simulations

    DEFF Research Database (Denmark)

    Brodersen, Mark Laier; Ou, Ge; Høgsberg, Jan Becker

    2016-01-01

    Results from real time hybrid simulations are compared to full numerical simulations for a hybrid viscous damper, composed of a viscous dashpot in series with an active actuator and a load cell. By controlling the actuator displacement via filtered integral force feedback the damping performance...... of the hybrid viscous damper is improved, while for pure integral force feedback the damper stroke is instead increased. In the real time hybrid simulations viscous damping is emulated by a bang-bang controlled Magneto-Rheological (MR) damper. The controller activates high-frequency modes and generates drift...... in the actuator displacement, and only a fraction of the measured damper force can therefore be used as input to the investigated integral force feedback in the real time hybrid simulations....

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

    Science.gov (United States)

    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....

  12. Establishment and application of SYBR Green Ⅰ real-time fluoresent quantitative RT-PCR assay for detection chicken interluekin-2 mRNA%鸡IL-2 mRNA SYBR Green Ⅰ实时荧光定量RT-PCR检测方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    王衡; 刘博奇; 宁章勇

    2013-01-01

    根据GenBank提供的ChIL-2参考序列设计了特异性引物,并以鸡3-磷酸甘油脱氢酶(GAPDH)基因为内参,以二者标准质粒为模板,应用SYBR Green Ⅰ实时荧光定量PCR技术,并采用双标准曲线相对定量方法建立了ChIL-2 mRNA荧光定量RT-PCR检测方法.结果显示,该方法该可以在3h左右对低拷贝量的模板(200 copy/μL)进行检测,同时该检测方法具有很好的特异性和重复性.雏鸡免疫试验IL-2 mRNA检测结果显示,该方法可有效应用于鸡体内IL-2在核酸水平的检测,并为今后IL-2相对定量的检测研究提供参考依据.%For establishing a method to detect chicken interleukin 2 (ChIL-2) mRNA expression,the primers were desigend for developing a SYBR Green Ⅰ real-time fluoresence quantitative RTPCR method,according to the chicken ChIL-2 gene and chicken glyceraldehyde-3-phosphate dehydrogenase (ChGAPDH) gene sequences available in GenBank of which plasmids were used as the templates.ChGAPDH was used as the internal control and "two standard curve relative quantitative" method was applied to compute the value.The results of this expriment revealed that this method could detect the template of low copies (200 copy/μL) in short time (about 3 hours),and also had a good specificity and repeatability.This method was used to detect the dynamic change of IL-2 mRNA in chicks which were inoculated with IBV S1 gene vaccine.The results of significant change between inoculated group and control group showed that this method could be effectively applied to detect the nucleic acid level of IL-2 in the body of chicken and provided a suitable reference approach for further research on relative quantification of ChIL-2.

  13. 双酚A诱导斑马鱼P53基因CDS突变的RT-PCR及序列分析%Bisphenol A induced zebrafish p53 gene CDS mutation of RT-PCR and sequence analysis

    Institute of Scientific and Technical Information of China (English)

    夏勇; 朱涵平; 董小岚; 傅剑云; 徐小民; 邹艳

    2011-01-01

    Objective :To study the mutagenic effect of bisphenol A (BPA) on zebrafish p53 gene coding sequence (CDS) in liver cells. Methods: 20 zebra fish were randomly divided into blank control group and exposed groups , each containing 10 fish.After exposing for 30 days of BPA, the RNA was extracted from Zebrafish liver tissue, the p53 gene including a complete coding sequence of was obtained by RT -PCR. Results: Sequence analysis showed that the P53 gene CDS length of 1122bp, encoding 373 amino acids, the percent identity between the published zebrafish sequence of p53 and ours' was 99.9% ,with the other biological sequence of p53 existing some differences. After 30 days exposure, BPA exposure group zebrafish p53 gene had mutated at the concentration of 0.6 mg/L. The base substitution of GAG→GGG at codon 135,AGG→CGG at codon 311 were detected by PCR - directed sequencing. This may result in the Glu→Gly of expressed p53 protein. Conclusion: The results showed that BPA on zebrafish p53 gene CDS is mutagenicity and using RT - PCR detection of p53 mutations induced by BPA is feasible.%目的:研究双酚A(bisphenol A,BPA)对斑马鱼肝脏p53基因编码序列(CDS)的致突变作用.方法:试验设空白对照组与BPA暴露组,连续染毒30 d后,从斑马鱼肝脏组织中提取RNA,通过RT-PCR方法扩增得到有完整编码区序列的p53基因,并进行测序分析.结果:序列分析表明,斑马鱼p53基因编码区序列长1122 bp,编码373个氨基酸,与网上已经登陆的斑马鱼p53序列有99.9%的相似系数,而与其他生物的p53序列存在一定的差异.在0.6 mg/L浓度下染毒30 d后,BPA暴露组斑马鱼p53基因序列发生了突变.直接测序证明,它们分别在135位、311位密码子发生GAG→GGG、AGG→CGG转变,使其编码的p53蛋白发生Glu→Gly变异.结论:试验结果表明BPA对斑马鱼p53基因CDS有致突变作用,采用RT-PCR技术检测BPA诱导p53基因突变是可行的.

  14. Eye tracker uncertainty analysis and modelling in real time

    Science.gov (United States)

    Fornaser, A.; De Cecco, M.; Leuci, M.; Conci, N.; Daldoss, M.; Armanini, A.; Maule, L.; De Natale, F.; Da Lio, M.

    2017-01-01

    Techniques for tracking the eyes took place since several decades for different applications that range from military, to education, entertainment and clinics. The existing systems are in general of two categories: precise but intrusive or comfortable but less accurate. The idea of this work is to calibrate an eye tracker of the second category. In particular we have estimated the uncertainty both in nominal and in case of variable operating conditions. We took into consideration different influencing factors such as: head movement and rotation, eyes detected, target position on the screen, illumination and objects in front of the eyes. Results proved that the 2D uncertainty can be modelled as a circular confidence interval as far as there is no stable principal directions in both the systematic and the repeatability effects. This confidence region was also modelled as a function of the current working conditions. In this way we can obtain a value of the uncertainty that is a function of the operating condition estimated in real time opening the field to new applications that reconfigure the human machine interface as a function of the operating conditions. Examples can range from option buttons reshape, local zoom dynamically adjusted, speed optimization to regulate interface responsiveness, the possibility to take into account the uncertainty associated to a particular interaction. Furthermore, in the analysis of visual scanning patterns, the resulting Point of Regard maps would be associated with proper confidence levels thus allowing to draw accurate conclusions. We conducted an experimental campaign to estimate and validate the overall modelling procedure obtaining valid results in 86% of the cases.

  15. Analysis and Optimization of Distributed Real-Time Embedded Systems

    DEFF Research Database (Denmark)

    Pop, Paul; Eles, Petru; Peng, Zebo

    2006-01-01

    An increasing number of real-time applications are today implemented using distributed heterogeneous architectures composed of interconnected networks of processors. The systems are heterogeneous not only in terms of hardware and software components, but also in terms of communication protocols...

  16. Analysis and optimisation of heterogeneous real-time embedded systems

    DEFF Research Database (Denmark)

    Pop, Paul; Eles, Petru; Peng, Zebo

    2006-01-01

    An increasing number of real-time applications are today implemented using distributed heterogeneous architectures composed of interconnected networks of processors. The systems are heterogeneous, not only in terms of hardware components, but also in terms of communication protocols and schedulin...

  17. Prognostic Value of RT-PCR Tyrosinase Detection in Peripheral Blood of Melanoma Patients

    Directory of Open Access Journals (Sweden)

    Esmeralda Carrillo

    2006-01-01

    Full Text Available Malignant melanoma (MM prognosis has been related to tumour thickness and clinical stage and metastasis risk has been associated with presence of tumour cells in peripheral blood. The aim of this study was to determine the relationship between presence of tyrosinase in peripheral blood of MM patients and their clinical prognosis. Blood samples from 58 MM patients (stage I–IV were analysed, using RT-PCR assay to detect tyrosinase mRNA. The results showed that positive RT-PCR assay for tyrosinase were significantly associated with clinical status and tumour thickness. After a median follow-up of 24 months, RT-PCR results were found to be significant correlated with recurrence (p < 0.05 and clinical stage III (p < 0.05. Separate analysis of stage III tumours to determine the prognostic value of tyrosinase presence in peripheral blood showed an overall 24-month survival rate of 70% in the RT-PCR negative group versus 10% in the positive group (p < 0.02. These results suggest that detection of circulating melanoma cells may be especially relevant in stage III patients, in whom RT-PCR positivity defines a subpopulation at high risk of recurrence.

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

    Science.gov (United States)

    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...

  19. Monitoring AML1-ETO mRNA levels by real-time quantitative RT-PCR in t (8; 21) acute myeloid leukemia patients after hematopoietic stem cell transplantation%实时定量RT-PCR方法监测急性髓系白血病患者造血干细胞移植后AML1-ETO融合基因mRNA水平的临床意义

    Institute of Scientific and Technical Information of China (English)

    王志东; 秦亚溱; 刘艳荣; 许兰平; 刘代红; 刘开彦; 黄晓军

    2008-01-01

    目的 评价实时定量RT-PCR(Q-PCR)方法监测AML1-ETO(+)急性髓系白血病(AML)患者异基因造血干细胞移植(allo-HSCT)后AML1-ETO mRNA水平的表达及其临床意义.方法 采用基于TagMan探针的Q-PCR技术检测17例AML1-ETO(+)AML患者allo-HSCT后不同时间骨髓标本AML1-ETO mRNA的表达.AML1-ETO mRNA水平以内参基因abl进行归一化.采用荧光原位杂交(FISH)法评估HSCT后是否达到细胞遗传学完全缓解(CCyR).结果 Q-PCR实验可重复敏感度为5个拷贝.在16例CCyR患者中,1例死于移植物抗宿主病(GVHD),1例死于感染,其余14例中位随访时间为268(70~811)d,HSCT后1个月(+1月)AML1-ETO中位水平0(0~0.740),+2月为0.026(0~2.900),+3月为0.039(0~3.300).移植时间超过12个月的5例患者中,中位随访685(385~811)d,4例仍呈AML1-ETO阳性,中位值0.078(0.003~0.120).1例复发患者+1月为0,+2月为9.800,+3月为5.600,+110 d血液学复发,AML1-ETO mRNA为390.000,+382 d死亡.结论 1年内AML1-ETO持续低水平阳性不一定预示复发;对AML1-ETO(+)AML患者HSCT后定期动态监测AML1-ETO水平十分必要.%Objective To evaluate the value of real time quantitative RT-PCR(Q-PCR) for monitoring AML1-ETO mRNA levels in AMLI-ETO(+) acute myeloid leukemia (AML) patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods Quantification of AMLI-ETO (+) mRNA was performed serially on bone marrow samples from 17 patients with AML1-ETO (+) AML after HSCT. Q-PCR used the TagMan probe system. The AML1-ETO mRNA level was normalized by control gene abl. Cytogenetic response was evaluated by fluorescent in situ hybridization (FISH). Results The reproducible sensitivity of Q-PCR was 5 copies. Out of 16 patients who achieved sustained complete cytogenetic response (CCyR), one each died of graft-versus-host disease and infection. The median AML1-ETO mRNA levels in the rest of 14 CCyR patients were 0 (0 - 0.740), 0. 026 (0 - 2.900), 0.039 (0 - 3.300) at

  20. Time-Stretch Accelerated Processor for Real-time, In-service, Signal Analysis

    DEFF Research Database (Denmark)

    Lonappan, Cejo K.; Buckley, Brandon W.; Adam, Jost

    2014-01-01

    We demonstrate real-time, in-service, digital signal analysis of 10 Gbit/s data using a 1.2 Tbit/s burst-mode digital processor. The processor comprises a time-stretch front-end and a custom data acquisition and real-time signal processing back- end. Experimental demonstration of real-time, in...

  1. Critical analysis of rhinovirus RNA load quantification by real-time reverse transcription-PCR.

    Science.gov (United States)

    Schibler, Manuel; Yerly, Sabine; Vieille, Gaël; Docquier, Mylène; Turin, Lara; Kaiser, Laurent; Tapparel, Caroline

    2012-09-01

    Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5'-untranslated regions (5'UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5'UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes

    Directory of Open Access Journals (Sweden)

    Mohamadhasan Tajadini

    2014-01-01

    Full Text Available Background: Real-time polymerase chain reaction (PCR is based on the revolutionary method of PCR. This technique is the result of PCR enormous sensitivity and real-time monitoring combination. In quantitative gene expression analysis, two methods have more popularity, SYBR Green and TaqMan, SYBR Green is relatively cost benefit and easy to use and technically based on binding the fluorescent dye to double-stranded deoxyribonucleic acid (dsDNA where TaqMan method has more expensive and based on dual labeled oligonucleotide and exonuclease activity of Taq polymerase enzyme. Specificity is the most important concern with the usage of any non-specific dsDNA-binding Dyes such as SYBR Green whiles more specificity showed by labeled oligonucleotide method such as TaqMan. In this study, we compared two common RT PCR methods, TaqMan and SYBR Green in measurement gene expression profile of adenosine receptors. Materials and Methods: Gene expression profiles of A1, A2A, A2B and A3 Adenosine receptors were analyzed by optimized TaqMan and SYBR Green quantitative RT PCR in breast cancer tissues. Primary expression data was normalizing by B. actin reference gene. Results: Efficiencies were calculated more than 95% for TaqMan and SYBR Green methods in all genes. The correlations between means of normalized data of each gene in two methods were positive and significant (P < 0.05. Conclusion: Data analysis showed that with the use of high performance primer and by use proper protocols and material we can make precise data by SYBR Green as TaqMan method. In other word by optimization of SYBR Green method, its performance and quality could be comparable to TaqMan method.

  5. A Real-Time Performance Analysis Model for Cryptographic Protocols

    Directory of Open Access Journals (Sweden)

    Amos Olagunju

    2012-12-01

    Full Text Available Several encryption algorithms exist today for securing data in storage and transmission over network systems. The choice of encryption algorithms must weigh performance requirements against the call for protection of sensitive data. This research investigated the processing times of alternative encryption algorithms under specific conditions. The paper presents the architecture of a model multiplatform tool for the evaluation of candidate encryption algorithms based on different data and key sizes. The model software was used to appraise the real-time performance of DES, AES, 3DES, MD5, SHA1, and SHA2 encryption algorithms.

  6. How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)

    Science.gov (United States)

    Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan

    Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

  7. Validation of tumor markers in central nervous system germ cell tumors by real-time reverse transcriptase polymerase chain reaction using formalin-fixed paraffin-embedded tissues.

    Science.gov (United States)

    Kim, Dowhan; Lee, Da Hye; Choi, Junjeong; Shim, Kyu Won; Kim, Se Hoon

    2013-01-01

    The therapeutic protocols for treatment of germinomas and non-germinomatous germ cell tumors (NGGCTs) are completely different, so it is important to distinguish pure germinomas from NGGCTs. As it can be difficult to diagnose by morphology alone, immunohisto-chemistry (IHC) has been widely used as an ancillary test to improve diagnostic accuracy. However, IHC has limitations due to the misinterpretation of results or the aberrant loss of immunoreactivity. However, real-time RT-PCR has certain advantages over IHC, including its quantitative nature. The aim of our study was to evaluate the usefulness of real-time RT-PCR on formalin-fixed paraffin-embedded (FFPE) tissue blocks for the diagnosis of germ cell tumors of the central nervous system. We selected eight markers of germ cell tumors using a literature search, and validated them using real-time RT-PCR. Among them, POU5F1, NANOG and TGFB2 were statistically significant (P=0.05) in multiple comparisons (MANOVA) of three groups (pure germinomas, mature teratomas and malignant germ cell tumors). Two-group (pure germinomas and NGGCTs) discriminant analysis achieved a 70.0% success rate in cross-validation. We concluded that real-time RT-PCR using FFPE tissue has adequate validating power comparable to IHC in the diagnosis of central nervous system germ cell tumors; therefore, when IHC is not available, not conclusive or not informative, RT-PCR is a potential alternative to a repeat biopsy.

  8. Testing and error analysis of a real-time controller

    Science.gov (United States)

    Savolaine, C. G.

    1983-01-01

    Inexpensive ways to organize and conduct system testing that were used on a real-time satellite network control system are outlined. This system contains roughly 50,000 lines of executable source code developed by a team of eight people. For a small investment of staff, the system was thoroughly tested, including automated regression testing, before field release. Detailed records were kept for fourteen months, during which several versions of the system were written. A separate testing group was not established, but testing itself was structured apart from the development process. The errors found during testing are examined by frequency per subsystem by size and complexity as well as by type. The code was released to the user in March, 1983. To date, only a few minor problems found with the system during its pre-service testing and user acceptance has been good.

  9. Real Time Analysis and Display of Aircraft Approach Maneuvers

    Science.gov (United States)

    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.

  10. Time-Stretch Accelerated Processor for Real-time, In-service, Signal Analysis

    DEFF Research Database (Denmark)

    Lonappan, Cejo K.; Buckley, Brandon W.; Adam, Jost

    2014-01-01

    We demonstrate real-time, in-service, digital signal analysis of 10 Gbit/s data using a 1.2 Tbit/s burst-mode digital processor. The processor comprises a time-stretch front-end and a custom data acquisition and real-time signal processing back- end. Experimental demonstration of real-time, in-service......, signal integrity analysis of streaming video packets at 10 Gbit/s is presented....

  11. RT-PCR detection of HIV in Republic of Macedonia.

    Science.gov (United States)

    Bosevska, Golubinka; Panovski, Nikola; Dokić, Eleni; Grunevska, Violeta

    2008-11-01

    The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works. The total of 33 examined persons were divided in two groups: 1) 13 persons seropositive for HIV; and 2) 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5). ELFA test for combined detection of HIV p24 antigen and anti HIV-1+2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity. Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly. In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70 masculineC, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20 masculineC for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is

  12. Real-Time Audio-Visual Analysis for Multiperson Videoconferencing

    Directory of Open Access Journals (Sweden)

    Petr Motlicek

    2013-01-01

    Full Text Available We describe the design of a system consisting of several state-of-the-art real-time audio and video processing components enabling multimodal stream manipulation (e.g., automatic online editing for multiparty videoconferencing applications in open, unconstrained environments. The underlying algorithms are designed to allow multiple people to enter, interact, and leave the observable scene with no constraints. They comprise continuous localisation of audio objects and its application for spatial audio object coding, detection, and tracking of faces, estimation of head poses and visual focus of attention, detection and localisation of verbal and paralinguistic events, and the association and fusion of these different events. Combined all together, they represent multimodal streams with audio objects and semantic video objects and provide semantic information for stream manipulation systems (like a virtual director. Various experiments have been performed to evaluate the performance of the system. The obtained results demonstrate the effectiveness of the proposed design, the various algorithms, and the benefit of fusing different modalities in this scenario.

  13. Real-Time Polymerase Chain Reaction: Applications in Diagnostic Microbiology

    Directory of Open Access Journals (Sweden)

    Kordo B. A. Saeed

    2013-11-01

    Full Text Available The polymerase chain reaction (PCR has revolutionized the detection of DNA and RNA. Real-Time PCR (RT-PCR is becoming the gold standard test for accurate, sensitive and fast diagnosis for a large range of infectious agents. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, high throughout and quantification. RT-PCR assays have advanced the diagnostic abilities of clinical laboratories particularly microbiology and infectious diseases. In this review we would like to briefly discuss RT-PCR in diagnostic microbiology laboratory, beginning with a general introduction to RT-PCR and its principles, setting up an RT PCR, including multiplex systems and the avoidance and remediation of contamination issues. A segment of the review would be devoted to the application of RT-PCR in clinical practice concentrating on its role in the diagnosis and treatment of infectious diseases.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. REAL TIME ANALYSIS OF WIRELESS CONTROLLER AREA NETWORK

    Directory of Open Access Journals (Sweden)

    Gerardine Immaculate Mary

    2014-09-01

    Full Text Available It is widely known that Control Area Networks (CAN are used in real-time, distributed and parallel processing which cover manufacture plants, humanoid robots, networking fields, etc., In applications where wireless conditions are encountered it is convenient to continue the exchange of CAN frames within the Wireless CAN (WCAN. The WCAN considered in this research is based on wireless token ring protocol (WTRP; a MAC protocol for wireless networks to reduce the number of retransmissions due to collision and the wired counterpart CAN attribute on message based communication. WCAN uses token frame method to provide channel access to the nodes in the system. This method allow all the nodes to share common broadcast channel by taken turns in transmitting upon receiving the token frame which is circulating within the network for specified amount of time. This method provides high throughput in bounded latency environment, consistent and predictable delays and good packet delivery ratio. The most important factor to consider when evaluating a control network is the end-to-end time delay between sensors, controllers, and actuators. The correct operation of a control system depends on the timeliness of the data coming over the network, and thus, a control network should be able to guarantee message delivery within a bounded transmission time. The proposed WCAN is modeled and simulated using QualNet, and its average end to end delay and packet delivery ratio (PDR are calculated. The parameters boundaries of WCAN are evaluated to guarantee a maximum throughput and a minimum latency time, in the case of wireless communications, precisely WCAN.

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

    Science.gov (United States)

    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.

  17. RT-PCR Protocols - Methods in Molecular Biology

    Directory of Open Access Journals (Sweden)

    Manuela Monti

    2011-03-01

    Full Text Available “The first record I have of it, is when I made a computer file which I usually did whenever I had an idea, that would have been on the Monday when I got back, and I called it Chain Reaction.POL, meaning polymerase. That was the identifier for it and later I called the thing the Polymerase Chain Reaction, which a lot of people thought was a dumb name for it, but it stuck, and it became PCR”. With these words the Nobel prize winner, Kary Mullis, explains how he named the PCR: one of the most important techniques ever invented and currently used in molecular biology. This book “RT-PCR Protocols” covers a wide range of aspects important for the setting of a PCR experiment for both beginners and advanced users. In my opinion the book is very well structured in three different sections. The first one describes the different technologies now available, like competitive RT-PCR, nested RT-PCR or RT-PCR for cloning. An important part regards the usage of PCR in single cell mouse embryos, stressing how important...........

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

    Directory of Open Access Journals (Sweden)

    Varinder Singh

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  20. Object oriented Full Function Point Analysis: A Model for Real Time Application

    Directory of Open Access Journals (Sweden)

    Sheeba Praveen

    2012-09-01

    Full Text Available this research work focused on determining the functional size of real time application at early stage. This paper will describe how to estimate the functional size of real time system using OO methodology. In this research we are proposing a Model which is very useful for estimating the size of real time system. And this model is fully based on Object oriented development Methodology due to the adaptation of the Function Point Analysis (FPA to Object Point Analysis (OPA. This model is for Real Time Application due to that have to take the FFP metric because FFP is best for size measurement of real time system. So the mapping is done in between Full Function Point metrics (FFP to Object Oriented Function Point metrics (OOFP and finally we are proposing a new metrics known as OOFFP and the based analysis is known as Object Oriented Full Function Point Analysis (OOFFPA.

  1. Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

    Institute of Scientific and Technical Information of China (English)

    Jing LU; Li QIN; Guang-jie LIU; Si-ting ZHAO; Xiao-ping CHEN

    2008-01-01

    Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus (SIV) infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction (RT-PCR). The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEM×174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA (b-DNA) assays or real-time PCR assays based on TaqMan probes.

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

    Directory of Open Access Journals (Sweden)

    Walton Eric F

    2007-10-01

    Full Text Available Abstract 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.

  3. A new StaRT-PCR approach to detect and quantify fish Viral Hemorrhagic Septicemia virus (VHSv): enhanced quality control with internal standards.

    Science.gov (United States)

    Pierce, Lindsey R; Willey, James C; Crawford, Erin L; Palsule, Vrushalee V; Leaman, Douglas W; Faisal, Mohamed; Kim, Robert K; Shepherd, Brian S; Stanoszek, Lauren M; Stepien, Carol A

    2013-04-01

    Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world's most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR(®) green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR(®) green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0×10(0)-1.2×10(5) VHSv/10(6)actb1 molecules in wild caught fishes and 1.0×10(0)-8.4×10(5) molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean=1.9×10(4)) than those without (1.1×10(3)) (p<0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv.

  4. Combination of immunohistochemistry, FISH and RT-PCR shows high incidence of Xp11 translocation RCC: comparison of three different diagnostic methods.

    Science.gov (United States)

    Lee, Hyun Jung; Shin, Dong Hoon; Noh, Gyu You; Kim, Young Keum; Kim, Ahrong; Shin, Nari; Lee, Jung Hee; Choi, Kyung Un; Kim, Jee Yeon; Lee, Chang Hun; Sol, Mee Young; Rha, Seo Hee; Park, Sung Woo

    2017-05-09

    We evaluated the frequency of translocation renal cell carcinoma (RCC) by reverse transcription polymerase chain reaction (RT-PCR) and how well the TFE3 immunoreactivity is concordant with TFE3 gene translocation status proved by fluorescence in situ hybridization (FISH) assay and RT-PCR. TFE3 and Cathepsin K expression was analyzed by immunohistochemistry in 185 RCC cases, and 48 cases either of more than weak expression of TFE3 or of positivity for Cathepsin K were done for FISH analysis and RT-PCR. All the RT-PCR positive cases were confirmed by cloning and sequencing. Of the 14 cases with strong nuclear TFE3 expression, 12 showed a break-apart signal by FISH. ASPL- and PRCC-TFE3 translocations were detected in 13 and one case, respectively, by RT-PCR. Of 21 cases with weak TFE3 expression, five were translocation-positive by FISH. ASPL-, PRCC-, and PSF-TFE3 translocations were detected by RT-PCR (n=3, 3, and 1, respectively). All 13 TFE3-negative/cathepsin K-positive cases were negative by FISH and two each harbored ASPL- and PRCC-TFE3 translocations that were detected by RT-PCR. A high rate of TFE3 immunoreactivity (8.6%) was confirmed by RT-PCR (13.5%) and FISH (9.7%). Higher translocation rate of RT-PCR means RT-PCR detected translocation in TFE3 weak expression group and only cathepsin K positive group more specifically than FISH. Thus, RT-PCR would complement FISH analysis for detecting translocation RCC with fusion partners.

  5. Human movement analysis with image processing in real time

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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%.

  7. Improved RT-PCR Assay to Quantitate the Pri-, Pre-, and Mature microRNAs with Higher Efficiency and Accuracy.

    Science.gov (United States)

    Tong, Li; Xue, Huihui; Xiong, Li; Xiao, Junhua; Zhou, Yuxun

    2015-10-01

    Understanding of the functional significance of microRNAs (miRNAs) requires efficient and accurate detection method. In this study, we developed an improved miRNAs quantification system based on quantitative real-time polymerase chain reaction (qRT-PCR). This method showed higher efficiency and accuracy to survey the expression of primary miRNAs (pri-miRNAs), precursor miRNAs (pre-miRNAs), and mature miRNAs. Instead of relative quantification method, we quantified the pri-miRNAs and pre-miRNAs with absolute qRT-PCR based on SYBR Green I fluorescence. This improvement corrected for the inaccuracy caused by the differences in amplicon length and PCR efficiency. We also used SYBR Green method to quantify mature miRNAs based on the stem-loop qRT-PCR method. We extended the pairing part of the stem-loop reverse transcript (RT) primer from 6 to 11 bp, which greatly increased the efficiency of reverse transcription PCR (RT-PCR). The performance of the improved RT primer was tested using synthetic mature miRNAs and tissue RNA samples. Results showed that the improved RT primer demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules.

  8. Exercise induced stress in horses: Selection of the most stable reference genes for quantitative RT-PCR normalization

    Directory of Open Access Journals (Sweden)

    Silvestrelli Maurizio

    2008-05-01

    Full Text Available Abstract Background Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare. In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR technologies to analyse the expression of candidate genes involved in the cellular stress response. Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. Results The expression of nine potential reference genes was evaluated in lymphocytes of ten endurance horses during strenuous exercise. These genes were tested by qRT-PCR and ranked according to the stability of their expression using three different methods (implemented in geNorm, NormFinder and BestKeeper. Succinate dehydrogenase complex subunit A (SDHA and hypoxanthine phosphoribosyltransferase (HPRT always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, transferrin receptor (TFRC and ribosomal protein L32 (RPL32 were constantly classified as the less reliable controls. Conclusion This study underlines the importance of a careful selection of reference genes for qRT-PCR studies of exercise induced stress in horses. Our results, based on different algorithms and analytical procedures, clearly indicate SDHA and HPRT as the most stable reference genes of our pool.

  9. Culture and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Proven Mycobacterium Tuberculosis Endophthalmitis: A Case Series.

    Science.gov (United States)

    Rishi, Ekta; Rishi, Pukhraj; Therese, K Lily; Ramasubban, Gayathri; Biswas, Jyotirmay; Sharma, Tarun; Bhende, Pramod; Susvar, Pradeep; Agarwal, Mamta; George, Amala Elizabeth; Delhiwala, Kushal; Sharma, Vishal Rajan

    2016-09-06

    To report early confirmation of Mycobacterium tuberculosis (MTB) endophthalmitis by detection of 85B mRNA in vitreous by a reverse transcriptase polymerase chain reaction (RT-PCR) technique. Retrospective, interventional case series of 5 patients with MTB endogenous endophthalmitis. Vitreous aspirate was subjected to Ziehl-Neelsen (ZN) staining, BACTEC MicroMGIT culture, RT-PCR targeting the 85B gene, real-time PCR targeting the IS6110 region, and nested PCR targeting the MPB64 gene and IS6110 region. Correlation between detection of MTB RNA, culture positivity, and ZN staining was studied. Five patients with endophthalmitis with no history of tuberculosis revealed acid-fast bacilli on ZN staining of vitreous. RT-PCR detected 85B RNA within 24 h. Culture for MTB was positive in 3/5 patients after 1 month. None of the eyes recovered any useful vision. RT-PCR can detect viable MTB RNA and provide evidence of active infection much earlier than culture.

  10. Development of an optimized method for the detection of airborne viruses with real-time PCR analysis

    Directory of Open Access Journals (Sweden)

    Legaki Euaggelia

    2011-07-01

    Full Text Available Abstract Background Airborne viruses remain one of the major public health issues worldwide. Detection and quantification of airborne viruses is essential in order to provide information regarding public health risk assessment. Findings In this study, an optimized new, simple, low cost method for sampling of airborne viruses using Low Melting Agarose (LMA plates and a conventional microbial air sampling device has been developed. The use of LMA plates permits the direct nucleic acids extraction of the captured viruses without the need of any preliminary elution step. Molecular detection and quantification of airborne viruses is performed using real-time quantitative (RT-PCR (Q(RT-PCR technique. The method has been tested using Adenoviruses (AdVs and Noroviruses (NoVs GII, as representative DNA and RNA viruses, respectively. Moreover, the method has been tested successfully in outdoor experiments, by detecting and quantifying human adenoviruses (HAdVs in the airborne environment of a wastewater treatment plant. Conclusions The great advantage of LMA is that nucleic acids extraction is performed directly on the LMA plates, while the eluted nucleic acids are totally free of inhibitory substances. Coupled with QPCR the whole procedure can be completed in less than three (3 hours.

  11. Integrating Real-Time Analysis With The Dendritic Cell Algorithm Through Segmentation

    CERN Document Server

    Gu, Feng; Aickelin, Uwe

    2010-01-01

    As an immune inspired algorithm, the Dendritic Cell Algorithm (DCA) has been applied to a range of problems, particularly in the area of intrusion detection. Ideally, the intrusion detection should be performed in real-time, to continuously detect misuses as soon as they occur. Consequently, the analysis process performed by an intrusion detection system must operate in real-time or near-to real-time. The analysis process of the DCA is currently performed offline, therefore to improve the algorithm's performance we suggest the development of a real-time analysis component. The initial step of the development is to apply segmentation to the DCA. This involves segmenting the current output of the DCA into slices and performing the analysis in various ways. Two segmentation approaches are introduced and tested in this paper, namely antigen based segmentation (ABS) and time based segmentation (TBS). The results of the corresponding experiments suggest that applying segmentation produces different and significantl...

  12. Real Time Metrics and Analysis of Integrated Arrival, Departure, and Surface Operations

    Science.gov (United States)

    Sharma, Shivanjli; Fergus, John

    2017-01-01

    A real time dashboard was developed in order to inform and present users notifications and integrated information regarding airport surface operations. The dashboard is a supplement to capabilities and tools that incorporate arrival, departure, and surface air-traffic operations concepts in a NextGen environment. As trajectory-based departure scheduling and collaborative decision making tools are introduced in order to reduce delays and uncertainties in taxi and climb operations across the National Airspace System, users across a number of roles benefit from a real time system that enables common situational awareness. In addition to shared situational awareness the dashboard offers the ability to compute real time metrics and analysis to inform users about capacity, predictability, and efficiency of the system as a whole. This paper describes the architecture of the real time dashboard as well as an initial set of metrics computed on operational data. The potential impact of the real time dashboard is studied at the site identified for initial deployment and demonstration in 2017; Charlotte-Douglas International Airport. Analysis and metrics computed in real time illustrate the opportunity to provide common situational awareness and inform users of metrics across delay, throughput, taxi time, and airport capacity. In addition, common awareness of delays and the impact of takeoff and departure restrictions stemming from traffic flow management initiatives are explored. The potential of the real time tool to inform the predictability and efficiency of using a trajectory-based departure scheduling system is also discussed.

  13. Selection and validation of appropriate reference genes for quantitative real-time PCR analysis of gene expression in Lycoris aurea

    Directory of Open Access Journals (Sweden)

    Rui eMa

    2016-04-01

    Full Text Available 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 sequencing (RNA

  14. Detection of colon flora in peritoneal drain fluid after colorectal surgery: can RT-PCR play a role in diagnosing anastomotic leakage?

    Directory of Open Access Journals (Sweden)

    P Willemsen

    2009-12-01

    Full Text Available Background and objectives: A semi-quantitative Real-Time PCR strategy was developed to identify indicator organisms for an indication on anastomotic leakage in peritoneal drainage fluid, Escherichia coli and Enterococcus faecalis."nMaterials and Methods: The analytical performance of the amplification method was validated with 10 culture-positive and 7 culture negative peritoneal drain fluid samples, obtained from 9 different patients with a colorectal anastomosis."nResults: Real-Time PCR results were fully concordant with the microbiological culture results. However, among the culture negative samples, four false-positive RT-PCR results were found. All false-positives originated from a single patient with a surgical site infection. This may indicate an elevated sensitivity of the RT-PCR method."nConclusion: The results showed that the semi-quantitative RT-PCR method has the clear potential to be useful as a powerful tool in early detection of anastomotic leakage.

  15. Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients

    Institute of Scientific and Technical Information of China (English)

    Aikaterini; Tsouma; Chrysanthi; Aggeli; Panagiotis; Lembessis; George; N; Zografos; Dimitris; P; Korkolis; Dimitrios; Pectasides; Maria; Skondra; Nikolaos; Pissimissis; Anastasia; Tzonou; Michael; Koutsilieris

    2010-01-01

    AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved det...

  16. Real-time quantitative reverse transcription-PCR analysis of expression stability of Actinobacillus pleuropneumoniae housekeeping genes during in vitro growth under iron-depleted conditions

    DEFF Research Database (Denmark)

    Nielsen, K. K.; Boye, Mette

    2005-01-01

    The aims of the present investigation were to develop and test a sensitive and reproducible method for the study of gene expression in the porcine lung pathogen Actinobacillus pleuropneumoniae by real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal...... up-regulation under iron-restricted conditions compared to bacteria grown in medium with sufficient iron. The observed expression patterns of the genes of interest were consistent with previous observations. This study therefore lends further support to the use of real-time quantitative RT...

  17. Cost Analysis for Real-time Java Scoped-memory Areas

    Directory of Open Access Journals (Sweden)

    Delvin Defoe

    2007-08-01

    Full Text Available Java has recently joined C and C++ as a development platform for real-time and embedded applications. Java's garbage collection, while generally a useful feature, can be problematic for these applications: garbage collection occurs at unpredictable times and its latency is typically unbounded. This can compromise necessary real-time guarantees. To overcome these limitations, the Real-Time for Java Expert Group (RTJEG proposed the Real-Time Specification for Java (RTSJ, which introduced new memory models and new threads to utilize those models. One such memory model uses scoped-memory areas, which work best in the context of a NoHeapRealtimeThread (NHRT. Although much work has been done with scoped-memory areas and NHRTs, there is no system-independent analysis of their costs. In this article we present an asymptotic analysis for RTSJ scoped-memory areas and NHRTs.

  18. Rapid identification viruses from nasal pharyngeal aspirates in acute viral respiratory infections by RT-PCR and electrospray ionization mass spectrometry.

    Science.gov (United States)

    Chen, Kuan-Fu; Rothman, Richard E; Ramachandran, Padmini; Blyn, Lawrence; Sampath, Rangarajan; Ecker, David J; Valsamakis, Alexandra; Gaydos, Charlotte A

    2011-04-01

    Diagnosis of the etiologic agent of respiratory viral infection relies traditionally on culture or antigen detection. This pilot evaluation compared performance characteristics of the RT-PCR and electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to conventional virologic methods for identifying multiple clinically relevant respiratory viruses in nasopharyngeal aspirates. The RT-PCR/ESI-MS respiratory virus surveillance kit was designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus. Patients (N=192) attending an emergency department during the 2007-2008 respiratory season consented, and "excess" frozen archived nasopharyngeal aspirates were analysed; 46 were positive by conventional virology and 69 by RT-PCR/ESI-MS, among which there were six samples with multiple viral pathogens detected. The sensitivity and specificity of the assay were 89.1% and 80.3%, respectively. Additional viruses that were not identified by conventional virology assays were detected (4 human bocaviruses and 7 coronaviruses). Samples in which the RT-PCR/ESI-MS results disagreed with conventional virology were sent for analysis by a third method using a commercial RT-PCR-based assay, which can identify viruses not detectable by conventional virologic procedures. Time to first result of RT-PCR/ESI-MS was 8h. RT-PCR/ESI-MS demonstrated capacity to detect respiratory viruses identifiable and unidentifiable by conventional methods rapidly.

  19. Analysis of Gene and Protein Expression in Atherosclerotic Mouse Aorta by Western Blot and Quantitative Real-Time PCR.

    Science.gov (United States)

    Rivera-Torres, José

    2015-01-01

    Atherosclerosis involves changes in gene and protein expression patterns in affected arteries. Quantification of these alterations is essential for understanding the molecular mechanisms underlying this pathology. Western blot and real-time PCR-used to quantify protein and messenger RNA levels, respectively-are invaluable molecular biology tools, particularly when material is limited. The availability of many genetically modified mouse models of atherosclerosis makes the mouse aorta an ideal tissue in which to carry out these expression pattern analyses. In this chapter, protocols are presented for mRNA and protein extraction from mouse aorta and for the accurate quantification of mRNA expression by RT-PCR and of proteins by western blot.

  20. Differential expression analysis of boron transporters and some stress-related genes in response to 24-epibrassinolide and boron by semi-quantitative RT-PCR in Arabidopsis thaliana (L. Heynh

    Directory of Open Access Journals (Sweden)

    Surgun Yonca

    2016-01-01

    Full Text Available Plant steroidal hormones, brassinosteroids (BRs, promote plant developmental processes and enhance tolerance to several abiotic stresses including high boron (B stress. To examine the possible role of BR in high B-induced stress at the transcriptional level, we investigated the response of B transporter genes (BOR1-4, high B-induced genes (MATE, Hsp-like, BR-induced genes (Hsp70-4, Hsp90-1 and other stress-related genes (LTI/COR78, LEA4-5 upon exogenous treatments of 24-epibrassinolide (EBL on Arabidopsis thaliana (L. Heynh exposed to high concentrations of boric acid (BA using semi-quantitative RT-PCR. BA treatments led to down regulation of BOR1 and BOR3 genes in leaf and root tissues and higher concentration of EBL further decreased expression of these genes in roots. The expression of high B-induced genes was observed to be upregulated by 1 μM EBL treatment under high B stress in both tissues of the seedlings. The upregulation of BR-induced genes were clearly evident in root tissues co-treated with 1 μM EBL and BA as compared to BA alone. Higher concentration of EBL was found to be more effective in increasing expression of LTI/COR78 gene in root and LEA4-5 gene in shoot tissues. To our knowledge, this is the first report how exogenous application of EBL modulates high B stress responses at molecular level in model plant Arabidopsis thaliana.

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

    Science.gov (United States)

    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.

  2. Phase-shifting Real-time Holographic Microscopy applied in micro-structures surface analysis

    Energy Technology Data Exchange (ETDEWEB)

    Brito, I V; Gesualdi, M R R [Universidade Federal do ABC, R. Santa Adelia 166, Bangu, Santo Andre, 09210-170, SP (Brazil); Muramatsu, M [Instituto de Fisica, Universidade de Sao Paulo, Rua do Matao, Travessa R 186, Cidade Universitaria, 05508-090, Sao Paulo, SP (Brazil); Ricardo, J, E-mail: isis.brito@ufabc.edu.br [Universidad de Oriente, Ave. Patricio Lumumba s/n, Santiago de Cuba (Cuba)

    2011-01-01

    The microscopic real-time analysis of micro structured materials is of great importance in various domains of science and technology. For other hand, the holographic interferometry comprises a group of powerful optical methods for non-destructive testing in surface analysis. The holographic microscopy uses the holographic interferometric techniques to obtain quantitative intensity and phase information of the optical waves by microscopic systems. With the development of CCD cameras, computers (hardware and software), and new materials for holographic recording, these techniques can be used to replace the classical form of registration and became promising tools in surface analysis. In this work, we developed a prototype of Photorefractive and Digital Holographic Microscope for real-time analysis of micro-structured systems based on the phase-shifting real-time holographic interferometry techniques. Using this apparatus, we are made analysis of shapes and surfaces to obtain the phase maps and the 3D profiles of some samples.

  3. A real-time system for biomechanical analysis of human movement and muscle function.

    Science.gov (United States)

    van den Bogert, Antonie J; Geijtenbeek, Thomas; Even-Zohar, Oshri; Steenbrink, Frans; Hardin, Elizabeth C

    2013-10-01

    Mechanical analysis of movement plays an important role in clinical management of neurological and orthopedic conditions. There has been increasing interest in performing movement analysis in real-time, to provide immediate feedback to both therapist and patient. However, such work to date has been limited to single-joint kinematics and kinetics. Here we present a software system, named human body model (HBM), to compute joint kinematics and kinetics for a full body model with 44 degrees of freedom, in real-time, and to estimate length changes and forces in 300 muscle elements. HBM was used to analyze lower extremity function during gait in 12 able-bodied subjects. Processing speed exceeded 120 samples per second on standard PC hardware. Joint angles and moments were consistent within the group, and consistent with other studies in the literature. Estimated muscle force patterns were consistent among subjects and agreed qualitatively with electromyography, to the extent that can be expected from a biomechanical model. The real-time analysis was integrated into the D-Flow system for development of custom real-time feedback applications and into the gait real-time analysis interactive lab system for gait analysis and gait retraining.

  4. Detection of let-7a microRNA by real-time PCR in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Hong-He Zhang; Xian-Jun Wang; Guo-Xiong Li; En Yang; Ning-Min Yang

    2007-01-01

    AIM:To establish an accurate and rapid stem-loop reverse transcriptional real-time PCR (RT-PCR) method to quantify human let-7a miRNA in gastric cancer.METHODS: According to the sequence of let-7a miRNA,the stem-loop reverse transcriptional primer, the primers and quantitative MGB probes of real-time PCR were designed and synthesized. The dynamic range and the sensitivity of quantitative reverse transcriptional real-time PCR were determined. The levels of let-7a miRNA were examined in 32 gastric carcinoma samples by stem-loop RT-PCR method.RESULTS: The dynamic range and sensitivity of the let-7a miRNA quantification scheme were evaluated,the result showed the assay could precisely detect 10copies of mature let-7a miRNA in as few as 0.05 ng of total RNA of gastric mucosa. The results of specificity analysis showed no fluorescence signal occurred even though 50 ng of human genomic DNA was added to the reverse transcription (RT) reaction. The expression level of let-7a miRNA in gastric tumor tissues was significantly lower compared to normal tissues in 14 samples from 32patients.CONCLUSION: The stem-loop RT-PCR is a reliable method to detect let-7a miRNA which may play an important role in the development of gastric carcinoma.

  5. Artificial neural networks for instantaneous analysis of real-time Rutherford backscattering spectra

    Energy Technology Data Exchange (ETDEWEB)

    Demeulemeester, J., E-mail: Jelle.Demeulemeester@fys.kuleuven.b [Instituut voor Kern- en Stralingsfysica and INPAC, K.U. Leuven, Celestijnenlaan 200D, B-3001 Leuven (Belgium); Smeets, D. [Instituut voor Kern- en Stralingsfysica and INPAC, K.U. Leuven, Celestijnenlaan 200D, B-3001 Leuven (Belgium); Barradas, N.P. [Instituto Tecnologico e Nuclear, Estrada Nacional 10, Apartado 21, 2686-953 Sacavem (Portugal); Centro de Fisica Nuclear da Universidade de Lisboa, Av. Prof. Gama Pinto 2, 1699 Lisboa Codex (Portugal); Vieira, A. [Instituto Superior de Engenharia do Porto, Rua S. Tome, 4200 Porto (Portugal); Comrie, C.M. [Department of Physics, University of Cape Town, Rondebosch 7700 (South Africa); Temst, K.; Vantomme, A. [Instituut voor Kern- en Stralingsfysica and INPAC, K.U. Leuven, Celestijnenlaan 200D, B-3001 Leuven (Belgium)

    2010-05-15

    This paper reports on the advantage of using artificial neural networks (ANNs) to analyze large sets of real-time Rutherford backscattering spectrometry (RBS) data. Real-time RBS, i.e. collecting RBS spectra at periodic time intervals during a thermal treatment, probes the full response of a thin film to the annealing in situ. Although very valuable insights can be gained by this technique, the time-consuming analysis of the vast amount of RBS spectra acquired during real-time RBS measurements has so far prevented the widespread use of real-time RBS. Setting up an ANN is quite an intensive process as well, but once trained, these ANNs can handle the analysis of large data sets practically instantaneously. As such, the beneficial combination of real-time RBS and ANN analysis forms a perfect synergy. In this test case, a network was trained and applied to analyze the Ni silicide growth during annealing of a thin 80 nm Ni film on Si(1 0 0). The ANN performance was validated by comparing the ANN results with the conventional analysis performed on the same data set.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Xiaoping eNiu

    2015-10-01

    Full Text Available 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 3 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.

  8. Real-Time Grouting Monitoring and Visualization Analysis System for Dam Foundation Curtain Grouting

    Institute of Scientific and Technical Information of China (English)

    樊贵超; 钟登华; 任炳昱; 崔博; 李晓超; 岳攀

    2016-01-01

    A real-time monitoring and 3D visualization analysis system is proposed for dam foundation curtain grouting. Based on the real-time control technology, the optimization method and the set theory, a mathematical model of the system is established. The real-time collection and transmission technology of the grouting data pro-vides a data foundation for the system. The real-time grouting monitoring and dynamic alarming method helps the system control the grouting quality during the grouting process, thus, the abnormalities of grouting, such as jacking and hydraulic uplift, can be effectively controlled. In addition, the 3D grouting visualization analysis technology is proposed to establish the grouting information model(GIM). The GIM provides a platform to visualize and ana-lyze the grouting process and results. The system has been applied to a hydraulic project of China as a case study, and the application results indicate that the real-time grouting monitoring and 3D visualization analysis for the grouting process can help engineers control the grouting quality more efficiently.

  9. Real-time forensic DNA analysis at a crime scene using a portable microchip analyzer.

    Science.gov (United States)

    Liu, Peng; Yeung, Stephanie H I; Crenshaw, Karin A; Crouse, Cecelia A; Scherer, James R; Mathies, Richard A

    2008-09-01

    An integrated lab-on-a-chip system has been developed and successfully utilized for real-time forensic short tandem repeat (STR) analysis. The microdevice comprises a 160-nL polymerase chain reaction reactor with an on-chip heater and a temperature sensor for thermal cycling, microvalves for fluidic manipulation, a co-injector for sizing standard injection, and a 7-cm-long separation channel for capillary electrophoretic analysis. A 9-plex autosomal STR typing system consisting of amelogenin and eight combined DNA index system (CODIS) core STR loci has been constructed and optimized for this real-time human identification study. Reproducible STR profiles of control DNA samples are obtained in 2h and 30min with DNA required for a complete DNA profile is 100 copies. To critically evaluate the capabilities of our portable microsystem as well as its compatibility with crime scene investigation processes, real-time STR analyses were carried out at a mock crime scene prepared by the Palm Beach County Sheriff's Office (PBSO). Blood stain sample collection, DNA extraction, and STR analyses on the portable microsystem were conducted in the field, and a successful "mock" CODIS hit was generated on the suspect's sample within 6h. This demonstration of on-site STR analysis establishes the feasibility of real-time DNA typing to identify the contributor of probative biological evidence at a crime scene and for real-time human identification.

  10. Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation

    Directory of Open Access Journals (Sweden)

    Schmid Michael

    2004-06-01

    Full Text Available Abstract Background Noroviruses (NoV have become one of the most commonly reported causative agents of large outbreaks of non-bacterial acute gastroenteritis worldwide as well as sporadic gastroenteritis in the community. Currently, reverse transcriptase polymerase chain reaction (RT-PCR assays have been implemented in NoV diagnosis, but improvements that simplify and standardize sample preparation, amplification, and detection will be further needed. The combination of automated sample preparation and real-time PCR offers such refinements. Methods We have designed a new real-time RT-PCR assay on the LightCycler (LC with SYBR Green detection and melting curve analysis (Tm to detect NoV RNA in patient stool samples. The performance of the real-time PCR assay was compared with that obtained in parallel with a commercially available enzyme immunoassay (ELISA for antigen detection by testing a panel of 52 stool samples. Additionally, in a collaborative study with the Baden-Wuerttemberg State Health office, Stuttgart (Germany the real-time PCR results were blindly assessed using a previously well-established nested PCR (nPCR as the reference method, since PCR-based techniques are now considered as the "gold standard" for NoV detection in stool specimens. Results Analysis of 52 clinical stool samples by real-time PCR yielded results that were consistent with reference nPCR results, while marked differences between the two PCR-based methods and antigen ELISA were observed. Our results indicate that PCR-based procedures are more sensitive and specific than antigen ELISA for detecting NoV in stool specimens. Conclusions The combination of automated sample preparation and real-time PCR provided reliable diagnostic results in less time than conventional RT-PCR assays. These benefits make it a valuable tool for routine laboratory practice especially in terms of rapid and appropriate outbreak-control measures in health-care facilities and other settings.

  11. Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis

    DEFF Research Database (Denmark)

    Andersen, Ulla Vestergaard; Haakansson, I. T.; Roust, Tina

    2013-01-01

    strongyle type parasites (NC1, NC2). SYBR Green Real-Time PCRs were performed in triplicates. Results revealed a statistically significant increase in PCR yield after three days, which was statistically associated with beginning embryonation of the eggs. In conclusion, storage time and developmental stage...... of strongyle eggs are significant sources of error in studies based on quantitative real-time PCR analysis. This study suggests that for refrigerated storage of more than three days, eggs should be inactivated and preserved for further analysis....

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

    Science.gov (United States)

    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

  13. Model-based schedulability analysis of safety critical hard real-time Java programs

    DEFF Research Database (Denmark)

    Bøgholm, Thomas; Kragh-Hansen, Henrik; Olsen, Petur

    2008-01-01

    In this paper, we present a novel approach to schedulability analysis of Safety Critical Hard Real-Time Java programs. The approach is based on a translation of programs, written in the Safety Critical Java profile introduced in [21] for the Java Optimized Processor [18], to timed automata models...... has been implemented in a tool, named SARTS, successfully used to verify the schedulability of a real-time sorting machine consisting of two periodic and two sporadic tasks. SARTS has also been applied on a number of smaller examples to investigate properties of our approach.......In this paper, we present a novel approach to schedulability analysis of Safety Critical Hard Real-Time Java programs. The approach is based on a translation of programs, written in the Safety Critical Java profile introduced in [21] for the Java Optimized Processor [18], to timed automata models...

  14. Analysis of the LTE Access Reservation Protocol for Real-Time Traffic

    DEFF Research Database (Denmark)

    Thomsen, Henning; Kiilerich Pratas, Nuno; Stefanovic, Cedomir;

    2013-01-01

    LTE is increasingly seen as a system for serving real-time Machine-to-Machine (M2M) communication needs. The asynchronous M2M user access in LTE is obtained through a two-phase access reservation protocol (contention and data phase). Existing analysis related to these protocols is based on the fo......LTE is increasingly seen as a system for serving real-time Machine-to-Machine (M2M) communication needs. The asynchronous M2M user access in LTE is obtained through a two-phase access reservation protocol (contention and data phase). Existing analysis related to these protocols is based...... of available data resources caused by variable load, and (2) detection of collisions in contention phase may not be possible. All of this affects transmission of real-time M2M traffic, where data packets have to be sent within a deadline and may have only one contention opportunity. We analyze the features...

  15. 芋花叶病毒的RT-PCR检测及外壳蛋白基因序列分析%The Detection of Dasheen mosaic virus by RT-PCR and Sequence Analysis of Its Coat Protein Gene

    Institute of Scientific and Technical Information of China (English)

    施世明; 王国平; 徐文兴; 王利平; 洪霓

    2012-01-01

    Reverse transcription PCR(RT-PCR)was used for the detection of Dasheen mosaic virus (DsMV)in taro[Colocasia esculenta(L.)Schott] from Hubei,Zhejiang and Shandong Provinces. Results revealed that the average viral infection frequency in 91 collected taro samples was 26.4%. RT-PCR products of 317 bp(covering partial coat protein gene)from 14 isolates were sequenced. Results showed that the obtained sequences had high intra-isolate nucleotide similarities,but inter-isolate nucleotide similarities were varied from 68.3% to 97.8%. The cp genes of two DsMV isolates named DsMV-SCS and DsMV-JH were sequenced,and their sizes were 951 bp and 987 bp,respectively. Their cp genes shared similarities of 79.0% at nucleotide(nt)level and 82.3% at amino acid(aa)level to each other and similarities of 73.0%–92.1% at nt level and 74.8%–98.2% at aa level to reported cp sequences of DsMV,respectively. The phylogenetic trees constructed based on nucleotide and deduced amino acid sequences of the cp of DsMV showed that isolates DsMV-SCS and DsMV-JH clustered into two different groups. There was no obvious correlation between phylogenetic positions and host or geographical origins of different DsMV isolates.%采用RT-PCR技术对采自中国湖北、浙江和山东的91份芋[Colocasia esculenta(L.)Schott]样品的芋花叶病毒(Dasheen mosaicvirus,DsMV)进行了检测,检出率为26.4%。对其中14个DsMV分离物的317bp扩增产物(为外壳蛋白基因的一部分)序列分析的结果显示,各分离物内的核苷酸变异相对较低,而分离物间存在较大的分子变异,相似性为68.3%~97.8%。对来自湖北和浙江的2个DsMV分离物DsMV-SCS和DsMV-JH的外壳蛋白基因(coatproteingene,cp)进行了测序,全长分别为951bp和987bp,二者cp核苷酸和氨基酸序列相似性分别为79.0%和82.3%,与已报道DsMV的cp核苷酸和氨基酸序列相似性分别为73.0%~92.1%和74.8%~98.2%,在构建的系统发育树上聚

  16. Flipping the analytical coin : closing the information flow loop in high speed (real time) analysis

    NARCIS (Netherlands)

    K.E. Shahroudi

    1997-01-01

    textabstractAnalysis modules tend to be set up as one way flow of information, i.e a clear distinction between cause and effect or input and output. However, as the speed of analysis approaches real time (or faster than movie rate), it becomes increasingly difficult for an external user to

  17. Flipping the analytical coin : closing the information flow loop in high speed (real time) analysis

    NARCIS (Netherlands)

    Shahroudi, K.E.

    1997-01-01

    Analysis modules tend to be set up as one way flow of information, i.e a clear distinction between cause and effect or input and output. However, as the speed of analysis approaches real time (or faster than movie rate), it becomes increasingly difficult for an external user to distinguish between c

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. Noninvasive Strategy Based on Real-Time in Vivo Cataluminescence Monitoring for Clinical Breath Analysis.

    Science.gov (United States)

    Zhang, Runkun; Huang, Wanting; Li, Gongke; Hu, Yufei

    2017-03-21

    The development of noninvasive methods for real-time in vivo analysis is of great significant, which provides powerful tools for medical research and clinical diagnosis. In the present work, we described a new strategy based on cataluminescence (CTL) for real-time in vivo clinical breath analysis. To illustrate such strategy, a homemade real-time CTL monitoring system characterized by coupling an online sampling device with a CTL sensor for sevoflurane (SVF) was designed, and a real-time in vivo method for the monitoring of SVF in exhaled breath was proposed. The accuracy of the method was evaluated by analyzing the real exhaled breath samples, and the results were compared with those obtained by GC/MS. The measured data obtained by the two methods were in good agreement. Subsequently, the method was applied to real-time monitoring of SVF in exhaled breath from rat models of the control group to investigate elimination pharmacokinetics. In order to further probe the potential of the method for clinical application, the elimination pharmacokinetics of SVF from rat models of control group, liver fibrosis group alcohol liver group, and nonalcoholic fatty liver group were monitored by the method. The raw data of pharmacokinetics of different groups were normalized and subsequently subjected to linear discriminant analysis (LDA). These data were transformed to canonical scores which were visualized as well-clustered with the classification accuracy of 100%, and the overall accuracy of leave-one-out cross-validation procedure is 88%, thereby indicating the utility of the potential of the method for liver disease diagnosis. Our strategy undoubtedly opens up a new door for real-time clinical analysis in a pain-free and noninvasive way and also guides a promising development direction for CTL.

  1. Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Osorio Yaneth

    2010-06-01

    Full Text Available Abstract Background The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules. Results Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, IL-13, IL-21, chemokine ligand (CCL 22, CCL17, Chemokine (C-C motif receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-ΔΔCt method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters. Conclusions The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.

  2. Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis

    Science.gov (United States)

    2010-01-01

    Background The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules. Results Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-ΔΔCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters. Conclusions The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections. PMID:20569429

  3. Development of a multiplex RT-PCR for simultaneous diagnosis of human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV) from clinical specimens.

    Science.gov (United States)

    Dayakar, Seetha; Pillai, Heera R; Thulasi, Vineetha P; Nair, Radhakrishnan R

    2016-12-01

    Human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV) are ubiquitous respiratory viral pathogens. They belong to the family Paramyxoviridae (subfamily Pneumovirinae) and is responsible for acute respiratory tract infections in children, elderly and immunocompromised patients. We designed and tested a multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) as a cost-effective alternative to real-time PCR and cell culture based detection for HMPV and HRSV. The newly developed PCR was used to screen nasal/throat swab samples from 356 patients with suspected acute respiratory infection attending the Government Medical College, Thiruvananthapuram, Kerala, India. The method was compared with a commercially available kit employing real time PCR, for its sensitivity and specificity. 53 (14.9 %) samples were positive for at least one tested pathogen by mRT-PCR. All except one among the positive samples showed similar pathogen profile when tested using real time PCR. 8 (15.1 %) among these 53 were positive for HRSVA, 33 (62.3 %) positive for HRSVB and 12 (22.6 %) were positive for HMPV. 17 (32.7 %) samples showed co-infections in them. Sensitivity and specificity of the mRT-PCR was comparable to that of the commercial kit. Our findings indicate that this newly developed mRT-PCR can be used as a cost-effective alternative for laboratory diagnosis of HMPV/HRSV infection and will significantly reduce diagnostic costs for these viruses in clinical settings.

  4. Evaluation of ALK gene rearrangement in central nervous system metastases of non-small-cell lung cancer using two-step RT-PCR technique.

    Science.gov (United States)

    Nicoś, M; Krawczyk, P; Wojas-Krawczyk, K; Bożyk, A; Jarosz, B; Sawicki, M; Trojanowski, T; Milanowski, J

    2017-05-22

    RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.

  5. Real-Time Shop-Floor Production Performance Analysis Method for the Internet of Manufacturing Things

    Directory of Open Access Journals (Sweden)

    Yingfeng Zhang

    2014-04-01

    Full Text Available Typical challenges that manufacturing enterprises are facing now are compounded by lack of timely, accurate, and consistent information of manufacturing resources. As a result, it is difficult to analyze the real-time production performance for the shop-floor. In this paper, the definition and overall architecture of the internet of manufacturing things is presented to provide a new paradigm by extending the techniques of internet of things (IoT to manufacturing field. Under this architecture, the real-time primitive events which occurred at different manufacturing things such as operators, machines, pallets, key materials, and so forth can be easily sensed. Based on these distributed primitive events, a critical event model is established to automatically analyze the real-time production performance. Here, the up-level production performance analysis is regarded as a series of critical events, and the real-time value of each critical event can be easily calculated according to the logical and sequence relationships among these multilevel events. Finally, a case study is used to illustrate how to apply the designed methods to analyze the real-time production performance.

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

    Directory of Open Access Journals (Sweden)

    Oliver A Müller

    Full Text Available 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.

  7. Real-time marker-free motion capture system using blob feature analysis

    Science.gov (United States)

    Park, Chang-Joon; Kim, Sung-Eun; Kim, Hong-Seok; Lee, In-Ho

    2005-02-01

    This paper presents a real-time marker-free motion capture system which can reconstruct 3-dimensional human motions. The virtual character of the proposed system mimics the motion of an actor in real-time. The proposed system captures human motions by using three synchronized CCD cameras and detects the root and end-effectors of an actor such as a head, hands, and feet by exploiting the blob feature analysis. And then, the 3-dimensional positions of end-effectors are restored and tracked by using Kalman filter. At last, the positions of the intermediate joint are reconstructed by using anatomically constrained inverse kinematics algorithm. The proposed system was implemented under general lighting conditions and we confirmed that the proposed system could reconstruct motions of a lot of people wearing various clothes in real-time stably.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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…

  10. [Near infrared spectroscopy and multivariate statistical process analysis for real-time monitoring of production process].

    Science.gov (United States)

    Wang, Yi; Ma, Xiang; Wen, Ya-Dong; Zou, Quan; Wang, Jun; Tu, Jia-Run; Cai, Wen-Sheng; Shao, Xue-Guang

    2013-05-01

    Near infrared diffusive reflectance spectroscopy has been applied in on-site or on-line analysis due to its characteristics of fastness, non-destruction and the feasibility for real complex sample analysis. The present work reported a real-time monitoring method for industrial production by using near infrared spectroscopic technique and multivariate statistical process analysis. In the method, the real-time near infrared spectra of the materials are collected on the production line, and then the evaluation of the production process can be achieved by a statistic Hotelling T2 calculated with the established model. In this work, principal component analysis (PCA) is adopted for building the model, and the statistic is calculated by projecting the real-time spectra onto the PCA model. With an application of the method in a practical production, it was demonstrated that a real-time evaluation of the variations in the production can be realized by investigating the changes in the statistic, and the comparison of the products in different batches can be achieved by further statistics of the statistic. Therefore, the proposed method may provide a practical way for quality insurance of production processes.

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

    Science.gov (United States)

    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…

  12. CyNC - towards a General Tool for Performance Analysis of Complex Distributed Real Time Systems

    DEFF Research Database (Denmark)

    Schiøler, Henrik; Jessen, Jan Jakob; Nielsen, Jens F. Dalsgaard

    2005-01-01

    The paper addresses the current state and the ongoing activities of a tool for performance analysis of complex real time systems. The tool named CyNC is based on network calculus allowing for the computation of backlogs and delays in a system from specified lower and upper bounds of external...

  13. DNA fragmentation of human sperm can be detected by ligation-mediated real-time polymerase chain reaction.

    Science.gov (United States)

    Lim, Jung Jin; Lee, Jin Il; Kim, Dong Hwan; Song, Seung-Hun; Kim, Hyung Joon; Lee, Woo Sik; Lee, Dong Ryul

    2013-12-01

    To determine whether ligation-mediated real-time polymerase chain reaction (LM-RT-PCR), which combines LM-PCR, and RT-PCR, can detect sperm DNA fragmentation (DF) in human semen samples. Three-way comparison of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), sperm chromatin dispersion (SCD), and LM-RT-PCR for detecting sperm DNA fragmentation. University hospital-based research laboratory. Twenty-five men presenting at an infertility clinic. Basic analysis of sperm concentration, motility, vitality, and morphology, with each semen sample equally divided into three aliquots that were evaluated for fragmentation using TUNEL, SCD, and LM-RT-PCR assays. In TUNEL and SCD assays, counts of the number of sperm with tetramethylrhodamine (TMR) red signals or no halo; in LM-RT-PCR results, evaluation of the threshold cycles (Ct) and relative fluorescence unit (RFU) values. The median percentage of sperm with positive results for fragmentation in the TUNEL and SCD assays were 20.5% and 20.7%, respectively. To compare the accuracy of the TUNEL, SCD, and LM-RT-PCR assays, we divided the semen samples into two groups according to the TUNEL results: low and high percentage of sperm fragmentation. In the LM-RT-PCR results, the values of the cycles of threshold (Ct) and relative fluorescence unit (RFU) statistically significantly differed between the low and high percentage of sperm fragmentation groups. Comparisons among the TUNEL, SCD, and LM-RT-PCR assays revealed that the correlation patterns according to DNA fragmentation were similar in both the groups with high and low percentage of DNA fragmentation. Our morphologic analysis indicated that the fragmentation of sperm DNA did not appear to influence sperm morphology. These results indicate that the LM-RT-PCR technique is another useful tool for detecting DNA fragmentation, a parameter of sperm quality in human semen alone or combined with TUNEL or SCD assays. Copyright © 2013 American Society for

  14. A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Dobrovic Alexander

    2010-12-01

    Full Text Available Abstract Background RNA extracted from formalin-fixed paraffin-embedded (FFPE samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA. Results We have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR assays. This single-tube quality control (QC assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon. Conclusions The multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.

  15. Methods for Preparation of MS2 Phage-Like Particles and Their Utilization as Process Control Viruses in RT-PCR and qRT-PCR Detection of RNA Viruses From Food Matrices and Clinical Specimens.

    Science.gov (United States)

    Mikel, P; Vasickova, P; Kralik, P

    2015-02-25

    RNA viruses are pathogenic agents of many serious infectious diseases affecting humans and animals. The detection of pathogenic RNA viruses is based on modern molecular methods, of which the most widely used methods are the reverse transcription polymerase chain reaction (RT-PCR) and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). All steps of RT-PCR and qRT-PCR should be strictly controlled to ensure the validity of obtained results. False-negative results may be caused not only by inhibition of RT or/and PCR steps but also by failure of the nucleic acid extraction step, particularly in the case of viral RNA extraction. The control of nucleic acid extraction generally involves the utilization of a non-pathogenic virus (process control virus) of similar structural properties to those of the target virus. Although in clinical samples the use of such process control virus is only recommended, in other kinds of settings such as food matrices its use is necessary. Currently, several different process control viruses are used for these purposes. Process control viruses can also be constructed artificially using technology for production of MS2 phage-like particles, which have many advantages in comparison with other used controls and are especially suited for controlling the detection and quantification of certain types of RNA viruses. The technology for production of MS2 phage-like particles is theoretically well established, uses the knowledge gained from the study of the familiar bacteriophage MS2 and utilizes many different approaches for the construction of the various process control viruses. Nevertheless, the practical use of MS2 phage-like particles in routine diagnostics is relatively uncommon. The current situation with regard to the use of MS2 phage-like particles as process control viruses in detection of RNA viruses and different methods of their construction, purification and use are summarized and discussed in this

  16. Development and application of a SYBR green RT-PCR for first line screening and quantification of porcine sapovirus infection.

    Science.gov (United States)

    Mauroy, Axel; Van der Poel, Wim H M; der Honing, Renate Hakze-Van; Thys, Christine; Thiry, Etienne

    2012-10-17

    Sapoviruses are single stranded positive sense RNA viruses belonging to the family Caliciviridae. The virus is detected in different species including the human and the porcine species as an enteric pathogen causing asymptomatic to symptomatic enteritis. In this study, we report the development of a rapid real time qRT-PCR based on SYBR Green chemistry for the diagnosis of porcine sapovirus infection in swine. The method allows the detection of porcine sapoviruses and the quantification of the genomic copies present in stool samples. During its development, the diagnostic tool showed good correlation compared with the gold standard conventional RT-PCR and was ten-fold more sensitive. When the method was applied to field samples, porcine noroviruses from genogroup 2 genotype 11b were also detected. The method was also applied to swine samples from the Netherlands that were positive for PoSaV infection. Phylogenetic results obtained from the samples showed that PoSaV sequences were genetically related to the currently described genogroup III, to the proposed genogroup VII and also to the MI-QW19 sequence (close to the human SaV sequences). A rapid, sensitive, and reliable diagnosis method was developed for porcine sapovirus diagnosis. It correlated with the gold standard conventional RT-PCR. Specificity was good apart for genogroup 2 genotype 11b porcine noroviruses. As a first line screening diagnosis method, it allows a quicker and easier decision on doubtful samples.

  17. Development and application of a SYBR green RT-PCR for first line screening and quantification of porcine sapovirus infection

    Directory of Open Access Journals (Sweden)

    Mauroy Axel

    2012-10-01

    Full Text Available Abstract Background Sapoviruses are single stranded positive sense RNA viruses belonging to the family Caliciviridae. The virus is detected in different species including the human and the porcine species as an enteric pathogen causing asymptomatic to symptomatic enteritis. In this study, we report the development of a rapid real time qRT-PCR based on SYBR Green chemistry for the diagnosis of porcine sapovirus infection in swine. Results The method allows the detection of porcine sapoviruses and the quantification of the genomic copies present in stool samples. During its development, the diagnostic tool showed good correlation compared with the gold standard conventional RT-PCR and was ten-fold more sensitive. When the method was applied to field samples, porcine noroviruses from genogroup 2 genotype 11b were also detected. The method was also applied to swine samples from the Netherlands that were positive for PoSaV infection. Phylogenetic results obtained from the samples showed that PoSaV sequences were genetically related to the currently described genogroup III, to the proposed genogroup VII and also to the MI-QW19 sequence (close to the human SaV sequences. Conclusions A rapid, sensitive, and reliable diagnosis method was developed for porcine sapovirus diagnosis. It correlated with the gold standard conventional RT-PCR. Specificity was good apart for genogroup 2 genotype 11b porcine noroviruses. As a first line screening diagnosis method, it allows a quicker and easier decision on doubtful samples.

  18. Respiratory virus multiplex RT-PCR assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections.

    Science.gov (United States)

    Deng, Jikui; Ma, Zhuoya; Huang, Wenbo; Li, Chengrong; Wang, Heping; Zheng, Yuejie; Zhou, Rong; Tang, Yi-Wei

    2013-04-01

    Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex II V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab (NPS) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children's Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system (Qiagen, Germany) and 17 respiratory viruses and genotypes including influenza A virus (FluA), FluB, parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), enteroviruses (EnV), human bocaviruses (hBoV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1), and FluA 2009 pandemic H1N1(H1N1-p) were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9-100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%-40.0%) were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H1N1-p and RSV (p=0.011-0.000). The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous

  19. Real-Time Analysis and Forecasting of Multisite River Flow Using a Distributed Hydrological Model

    Directory of Open Access Journals (Sweden)

    Mingdong Sun

    2014-01-01

    Full Text Available A spatial distributed hydrological forecasting system was developed to promote the analysis of river flow dynamic state in a large basin. The research presented the real-time analysis and forecasting of multisite river flow in the Nakdong River Basin using a distributed hydrological model with radar rainfall forecast data. A real-time calibration algorithm of hydrological distributed model was proposed to investigate the particular relationship between the water storage and basin discharge. Demonstrate the approach of simulating multisite river flow using a distributed hydrological model couple with real-time calibration and forecasting of multisite river flow with radar rainfall forecasts data. The hydrographs and results exhibit that calibrated flow simulations are very approximate to the flow observation at all sites and the accuracy of forecasting flow is gradually decreased with lead times extending from 1 hr to 3 hrs. The flow forecasts are lower than the flow observation which is likely caused by the low estimation of radar rainfall forecasts. The research has well demonstrated that the distributed hydrological model is readily applicable for multisite real-time river flow analysis and forecasting in a large basin.

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

    Science.gov (United States)

    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.

  1. Writing/Thinking in Real Time: Digital Video and Corpus Query Analysis

    Directory of Open Access Journals (Sweden)

    Park, Kwanghyun

    2010-10-01

    Full Text Available 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 learning. A perennial challenge to research using digital video (e.g., screen recordings has been the method for interfacing the captured behavior with the learners’ cognition. An exploratory proposal in this paper is that with an additional layer of data (i.e., corpus search queries, analyses of real-time data can be extended to provide an explicit representation of learner’s cognitive processes. This paper describes the method and applies it to an area of SLA, specifically writing, and presents an in-depth, moment-by-moment analysis of an L2 writer’s composing process. The findings show that the writer’s composing process is fundamentally developmental, and that it is facilitated in her dialogue-like interaction with an artifact (i.e., the corpus. The analysis illustrates the effectiveness of the method for capturing learners’ cognition, suggesting that L2 learning can be more fully explicated by interpreting real-time data in concert with investigation of corpus search queries.

  2. Desing and Real-Time Analysis of a Direct Current Motor Controller

    Directory of Open Access Journals (Sweden)

    Reinier Torres Labrada

    2011-10-01

    Full Text Available The real time analysis of a DC motor controller is addressed. During the functional analysis, deadline and period of tasks were extracted to be used as parameters for Response Time Analysis (RTA. To get the Worst Case Execution Time (WCET each task is stimulated with a random input vector. A great number of computation time samples were recorded and the WCET computed using the Gumbel statistical distribution. With the WCET, deadlines and periods of the task set under analysis a RTA model is evaluated and its results discussed. By modeling the WCET using the Extreme Values Theory (EVT the need of low level analysis for software and an accurate model for hardware is avoided. The method used here is useful for soft real-time systems and could be applied to other hw/sw platforms easily.

  3. Methodology for object-oriented real-time systems analysis and design: Software engineering

    Science.gov (United States)

    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.

  4. RT-PCR test for detecting porcine sapovirus in weanling piglets in Hunan Province, China.

    Science.gov (United States)

    Liu, Guo-Hua; Li, Run-Cheng; Huang, Ze-Bin; Yang, Jun; Xiao, Chao-Ting; Li, Jing; Li, Man-Xiang; Yan, Yun-Qiu; Yu, Xing-Long

    2012-10-01

    The prevalence of porcine sapovirus infection in weanling pigs was investigated in Hunan Province, China, between August 2006 and October 2007. A total of 153 diarrheic fecal samples from ten intensive pig farms from ten representative administrative regions in Hunan province were examined for porcine sapoviruses using RT-PCR. Twenty-two of 153 (14.37 %) samples were found to contain porcine sapoviruses. Phylogenetic analysis showed that all the porcine sapovirus isolates in Hunan Province belonged to the porcine sapovirus genogroup III. The results of the present investigation have implications for the control of porcine sapovirus infection in pigs in Hunan Province, China.

  5. PrimerSeq:Design and Visualization of RT-PCR Primers for Alternative Splicing Using RNA-seq Data

    Institute of Scientific and Technical Information of China (English)

    Collin Tokheim; Juw Won Park; Yi Xing

    2014-01-01

    The vast majority of multi-exon genes in higher eukaryotes are alternatively spliced and changes in alternative splicing (AS) can impact gene function or cause disease. High-throughput RNA sequencing (RNA-seq) has become a powerful technology for transcriptome-wide analysis of AS, but RT-PCR still remains the gold-standard approach for quantifying and validating exon splicing levels. We have developed PrimerSeq, a user-friendly software for systematic design and visualization of RT-PCR primers using RNA-seq data. PrimerSeq incorporates user-provided tran-scriptome profiles (i.e., RNA-seq data) in the design process, and is particularly useful for large-scale quantitative analysis of AS events discovered from RNA-seq experiments. PrimerSeq features a graphical user interface (GUI) that displays the RNA-seq data juxtaposed with the expected RT-PCR results. To enable primer design and visualization on user-provided RNA-seq data and transcript annotations, we have developed PrimerSeq as a stand-alone software that runs on local computers. PrimerSeq is freely available for Windows and Mac OS X along with source code at http://primerseq.sourceforge.net/. With the growing popularity of RNA-seq for transcriptome stud-ies, we expect PrimerSeq to help bridge the gap between high-throughput RNA-seq discovery of AS events and molecular analysis of candidate events by RT-PCR.

  6. A Macroscopic Mathematical Model for Cell Migration Assays Using a Real-Time Cell Analysis.

    Science.gov (United States)

    Di Costanzo, Ezio; Ingangi, Vincenzo; Angelini, Claudia; Carfora, Maria Francesca; Carriero, Maria Vincenza; Natalini, Roberto

    Experiments of cell migration and chemotaxis assays have been classically performed in the so-called Boyden Chambers. A recent technology, xCELLigence Real Time Cell Analysis, is now allowing to monitor the cell migration in real time. This technology measures impedance changes caused by the gradual increase of electrode surface occupation by cells during the course of time and provide a Cell Index which is proportional to cellular morphology, spreading, ruffling and adhesion quality as well as cell number. In this paper we propose a macroscopic mathematical model, based on advection-reaction-diffusion partial differential equations, describing the cell migration assay using the real-time technology. We carried out numerical simulations to compare simulated model dynamics with data of observed biological experiments on three different cell lines and in two experimental settings: absence of chemotactic signals (basal migration) and presence of a chemoattractant. Overall we conclude that our minimal mathematical model is able to describe the phenomenon in the real time scale and numerical results show a good agreement with the experimental evidences.

  7. A Novel Framework for Real-Time Fault Diagnosis Based on Dynamic Fault Tree Analysis

    Directory of Open Access Journals (Sweden)

    Rongxing Duan

    2013-02-01

    Full Text Available To meet the real-time diagnosis requirements of the complex system, this study proposes a novel framework for real-time fault diagnosis using dynamic fault tree analysis. It pays special attention to meeting two challenges: model development and real-time reasoning. In terms of the challenge of model development, we use a dynamic fault tree model to capture the dynamic behavior of system failure mechanisms and calculate some reliability results by mapping a dynamic fault tree into an equivalent Bayesian Network (BN in order to avoid the infamous state space explosion problem. In terms of the real-time reasoning challenge, we adopt a logic compilation based inference algorithm, which compiles the BN into an arithmetic circuit and retrieves answers to probabilistic queries by evaluating and differentiating the arithmetic circuit. Furthermore, we incorporate sensors data into fault diagnosis, cope with the sensors reliability and propose the schemes on how to update the Diagnostic Importance Factor (DIF and the minimal cut sets. Finally, a case study is given to validate the efficiency of this method.

  8. Timing Analysis of Mixed-Criticality Hard Real-Time Applications Implemented on Distributed Partitioned Architectures

    DEFF Research Database (Denmark)

    Marinescu, Sorin Ovidiu; Tamas-Selicean, Domitian; Acretoaie, Vlad;

    In this paper we are interested in the timing analysis of mixed-criticality embedded real-time applications mapped on distributed heterogeneous architectures. Mixedcriticality tasks can be integrated onto the same architecture only if there is enough spatial and temporal separation among them. We...... in partitions using fixedpriority preemptive scheduling. We have extended the stateof- the-art algorithms for schedulability analysis to take into account the partitions. The proposed algorithm has been evaluated using several synthetic and real-life benchmarks....

  9. One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

    Science.gov (United States)

    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.

  10. Development of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    Science.gov (United States)

    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.

  11. Genotyping of Rotavirus by Using RT-PCR Methods

    Directory of Open Access Journals (Sweden)

    Hera Nirwati

    2015-11-01

    Full Text Available There is a great diversity of rotavirus genotypes circulating worldwide, with dominant genotypes changing from year to year. Rotavirus genotyping was performed by using reverse transcription PCR with type-specifi c-primers. Since rotavirus is a RNA virus that has high mutation rate, there was a possibility of technical diffi culty in genotyping due to mutation in the primer binding sites. During Indonesian rotavirus surveillance study 2006-2009, it was reported that 17% of samples subjected for G type and 21% of samplessubjected for P type were untypeable. The objective of this study was to identify genotypes of the samples that were untypeable previously using RT-PCR based on the method described by Das et al. (1994 and Gentsch et al. (1992. There were 30 samples subjected to G type and 61 samples subjected to P type to be re-typed using method described by Gouvea et al. (1990 and Simmond et al. (2008 for G and P typing, respectively. By using another set of primer, the genotype of all samples was identifi ed. This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses.Key words: rotavirus, G typing, P typing

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

    Science.gov (United States)

    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.

  13. Real-time reverse transcriptase PCR for the rapid and sensitive detection of Salmonella typhimurium from pork.

    Science.gov (United States)

    Techathuvanan, Chayapa; Draughon, Frances Ann; D'Souza, Doris Helen

    2010-03-01

    Reverse transcriptase PCR (RT-PCR) detects the presence of mRNA and has a greater potential for detecting viable pathogens than do DNA-based PCR assays, with improved speed and sensitivity compared with traditional methods. Our objective was to rapidly and sensitively detect Salmonella Typhimurium from pork within two 8-h work shifts using a SYBR Green I real-time RT-PCR (rt-RT-PCR) assay. Pork chop and sausage samples (25 g) were inoculated with 10(8) to 10(0) CFU of Salmonella Typhimurium and stomached in 225 ml of tetrathionate broth. Serial dilutions were spread plated on xylose lysine Tergitol 4 agar either immediately or after 10 h of selective preenrichment or preenrichment followed by 12 h of selective enrichment (for stressed cells) at 37 degrees C for standard cultural enumeration. RNA was extracted using the TRIzol method. The rt-RT-PCR assay was carried out in a Bio-Rad iCycler using a SYBR Green I one-step RT-PCR kit and Salmonella specific invA gene primers with an internal amplification control (IAC). The PCR was followed by melting temperature (T(m)) analysis to determine specific Salmonella invA (T(m) = 87.5 degrees C) and IAC (T(m) = 82 degrees C) products. Improved Salmonella detection up to 10(1) CFU/25 g of pork and 10(0) CFU/25 g of sausages was obtained after 10 h of enrichment within approximately 24 h. Even without enrichment, Salmonella could be detected from both pork chop and sausage at 10(6) CFU/25 g within 1 day. This robust rt-RT-PCR detects and confirms Salmonella in pork within approximately 24 h and thus is significantly faster than traditional methods that take >/=1 week. This assay shows promise for routine testing and monitoring of Salmonella by the pork industry.

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

    Science.gov (United States)

    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.

  15. Establishment of realtime RT-PCR assay to detect polio virus in the Acute Flaccid Paralysis laboratory surveillance

    Directory of Open Access Journals (Sweden)

    Nike Susanti

    2016-07-01

    Full Text Available AbstrakLatar belakang: Virus polio indigenous terakhir ditemukan di Indonesia tahun 1995 tetapi ancaman viruspolio impor dan mutasi virus dari Oral Polio Vaccine (OPV menjadi Vaccine Derived Poliovirus (VDPVmasih berlanjut. Tahun 1991 WHO mengembangkan Surveilans Acute Flaccid Paralysis (AFP dan tahun2014, identifikasi virus polio dengan real-time reverse transcriptase Polymerase Chain Reaction (rRTPCRmulai digunakan di Laboratorium Nasional Polio Pusat Biomedis dan Teknologi Dasar Kesehatan.Tujuan dari penggunaan rRT-PCR untuk mendapatkan metode yang cepat dan lebih baik dalam memantausirkulasi dan mutasi virus polio.Metode: Isolat polio positif diidentifikasi menggunakanan rRT PCR dengan kombinasi primer dan probeyang ditetapkan WHO. RNA virus di konversi ke cDNA menggunakan reverse transcriptase lalu diamplifikasimenggunakan taq polymerase. Produk PCR di deteksi dan diidentifikasi dengan hibridisasi menggunakanprobe spesifik. Sintesis cDNA dan reaksi PCR menggunakan primer yang dilekatkan di probe. Kombinasiprimer dan probe menghasilkan identifikasi serotipe dan intratypic differentiation (ITD dari isolat virus.Hasil: Selama tahun 2014, NPL Jakarta menerima 604 kasus AFP dari surveilans dan lima kasusterdeteksi positif mengandung virus polio. Semua spesimen positif mengandung virus polio yang berasaldari vaksin. Dua kasus positif virus polio tipe P2 (40%, satu kasus jenis virus polio P1 (20%, 1 kasusjenis virus polio P3 (20% dan satu kasus virus polio campuran jenis P1 + P2 (20%.Kesimpulan: Real-time PCR dapat digunakan di Laboratorium Polio Jakarta untuk membantu identifikasivirus Polio secara cepat. Tes ini dapat digunakan untuk memantau sirkulasi virus polio pada populasiyang rutin diimunisasi dengan OPV. (Health Science Journal of Indonesia 2016;7:27-31Kata kunci: ITD, Poliovirus, Identification, rRT-PCR AbstractBackground: The last indigenous polio was detected in 1995 but the threat of wild type polio viruses and themutation of Oral

  16. Throughput Analysis for a High-Performance FPGA-Accelerated Real-Time Search Application

    Directory of Open Access Journals (Sweden)

    Wim Vanderbauwhede

    2012-01-01

    Full Text Available We propose an FPGA design for the relevancy computation part of a high-throughput real-time search application. The application matches terms in a stream of documents against a static profile, held in off-chip memory. We present a mathematical analysis of the throughput of the application and apply it to the problem of scaling the Bloom filter used to discard nonmatches.

  17. Real-time open-loop frequency response analysis of flight test data

    Science.gov (United States)

    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.

  18. Detection of bacterial species involved in perimplantitis concerned with cultural and RT-PCR

    Directory of Open Access Journals (Sweden)

    Marcello Gatti

    2010-06-01

    Full Text Available Dental implants offer new treatment options for edentulous either partially or completely, now represent a viable alternative to conventional fixed protheses. Dental implants are colonized by a flora dominated by Gram-positive facultative aerobic, while in patients with bone loss and formation of pockets peri-implant diseases was found a significant difference in the composition of microflora, bacteria, Gram-negative anaerobes in particular Fusobacterium spp., Treponema denticola (Spirochetes, Tannerella forsythensis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia as interim black-pigmented bacteria, Porphyromonas gingivalis, often in high concentrations. Aims. The purpose of this study was to identify those at risk of perimplantitis using 2 techniques: RT-PCR examination of trade and culture. The results were compared taking into consideration the advantages and disadvantages of both methods. Materials and methods.We studied 24 patients (14 women and 10 men, aged, women between 43 and 76 years, with an average of 63.8 + / - 10.9 years, men between 45 and 88 years with a average of 64.3 years + / - 12.5 years. Was performed a double levy of sub-gingival plaque at multiple sites that had an implant CAL (clinical attachment level> 4mm in order to assess