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Sample records for chain reaction rt-pcr

  1. Simultaneous detection of enteropathogenic viruses in buffalos faeces using multiplex reverse transcription-polymerase chain reaction (mRT-PCR

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    U. Pagnini

    2010-02-01

    Full Text Available A multiplex reverse transcription- polymerase chain reaction (mRT-PCR assay that detects Bovine Viral Diarrhoea Virus, Bovine Coronavirus, and Group A Rotaviruses in infected cell-culture fluids and clinical faecal samples is described. One hundred twenty faecal samples from buffalo calves with acute gastroenteritis were tested. The mRT-PCR was validated against simplex RT-PCR with published primers for Pestivirus, Coronavirus and Rotavirus. The multiplex RT-PCR was equally sensitive and specific in detecting viral infections compared with simplex RT-PCR. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. This mRT-PCR may be a sensitive and rapid assay for surveillance of buffalo enteric viruses in field specimens. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of acute gastroenteritis.

  2. A Comparative Analysis of Routine Techniques: Reverse Transcriptase Polymerase Chain Reaction (RT-PCR and Five Cell Lines for Detection of Enteroviruses in Stool Specimens

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    F Abbasian

    2011-06-01

    Full Text Available Background and objectives: Each year, Enteroviruses infect millions of people and cause different diseases. The agents are usually detected using cell culture. RD (Rhabdomyosarcoma and L20B (L cells are among the recommended cells by the World Health Organisation (WHO for this purpose. Even though cell culture is the most common method used in diagnosing Enteroviruses in stool specimens, this particular method poses some problems, which include false positive or negative results, lack of a unique cell line for diagnosing all Enterovirus types in addition to being time consuming. For these reasons, an attempt was made to find better techniques of Enterovirus detection. RT-PCR (Reverse Transcriptase Polymerase Chain Reaction is a technique used in place of the cell culture method. In this study, the cell culture method was compared with RT-PCR for detection of Enteroviruses in stool specimens.Material and method: First, the chloroform treated stool samples were inoculated onto five cell lines, including RD, L20B, Hep-2 (Human Epidermoid carcinoma cell line, Vero (Verde Reno and GMK (Green Monkey Kidney. The results were then compared with data from Enterovirus detection using the RT-PCR technique.Results and conclusion: The difference between RT-PCR and cell culture results was significant. Enteroviruses were detected in 24% of specimens using RT-PCR while cell lines could isolate Enteroviruses in just 14.4% of the samples.

  3. Use of spontaneously mutated human DNA as competitive internal standard for nucleic acid quantification by reverse transcription-polymerase chain reaction (RT-PCR)

    International Nuclear Information System (INIS)

    Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcription as well in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe a novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary 'epidermilysis bulosa dystrophica' associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by 'Ear I' endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of α32P-labelled dCTP. The reaction was followed by 'Ear I' digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR. (author). 12 refs, 3 figs

  4. Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR method

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    FIGUEIREDO Luiz Tadeu Moraes

    1997-01-01

    Full Text Available We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

  5. 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.; Hutchings, G.H.; King, D. P.; Ferris, N.P.; Alexandersen, Søren

    2004-01-01

    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......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...

  6. 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. PMID:24582581

  7. DEVELOPMENT OF HOMOLOGOUS VIRAL INTERNAL CONTROLS FOR USE IN RT-PCR ASSAYS OF WATERBORNE ENTERIC VIRUSES

    Science.gov (United States)

    Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides ...

  8. DETECTION SENSITIVITY AND QUANTITATION OF PLASMODIUM FALCIPARUM VAR GENE TRANSCRIPTS BY REAL-TIME RT-PCR IN COMPARISON WITH CONVENTIONAL RT-PCR

    OpenAIRE

    Gatton, Michelle L; Peters, Jennifer M.; GRESTY, KARRYN; Fowler, Elizabeth V.; Chen, Nanhua; Cheng, Qin

    2006-01-01

    Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there wa...

  9. RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza

    DEFF Research Database (Denmark)

    Dybkær, Karen; Munch, Mette; Handberg, Kurt; Jørgensen, Poul Henrik

    A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected with...... low-pathogenicity AIV, from wild aquatic birds, and from domestic ducks. The AIV was detected in 32 swab pools by RT-PCR-ELISA compared to 23 by virus isolation (VI) in embryonated specific pathogen free (SPF) chicken eggs. Thus, 39% more specimens were positive by RT-PCR-ELISA than by VI. Two of the...

  10. Comparison of RT-PCR-Dot blot hybridization based on radioisotope 32P with conventional RT-PCR and commercial ELISA Assays for blood screening of HIV-1

    International Nuclear Information System (INIS)

    There are many commercial ELISA and rapid test kits that have been used for blood screening; however, the kits can give false positive and negative results. Therefore, RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Dot Blot Hybridization based on radioisotope 32P (RDBR) method was developed in this research, to compare the method with the conventional RT-PCR and commercial ELISA Enzyme-Linked lmmunosorbent Assay) kit. This method is efficient for screening of large blood specimens and surveillance study. Eighty seven samples were used and serum of the samples were tested by ELISA to detect HIV-1. The HIV-l RNA genome was extracted from plasma samples and tested using the RT-PCR and RDBR methods. Of 87 samples that were tested, the rates of positive testing of the RT-PCR, the RDBR, and the ELISA were 71.26%, 74.71%, and 80.46%, respectively. The RDBR (a combination of RTPCR and dot blot hybridization) was more sensitive than conventional RT-PCR by showing 3.45% in increase number of positive specimens. The results showed that of 9 samples (10.34%) were negative RDBR and positive ELISA, while 4 samples (4.60%) were negative ELISA and positive RDBR. The two methods showed slightly difference in the results but further validation is still needed. However, RDBR has high potential as an alternative method for screening of blood in large quantities when compared to method of conventional RT-PCR and ELISA. (author)

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

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    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.

  12. Validation of putative reference genes for qRT-PCR normalization in tissues and blood from pigs infected with Actinobacillus pleuropneumoniae

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Mortensen, Shila; Poulsen, K.T.; Angen, Øystein; Heegaard, Peter M. H.

    The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive and very efficient technique for quantification of gene expression. However, qRT-PCR relies on accurate normalization of gene expression data, as RNA recovery and cDNA synthesis efficiency might va...

  13. 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 ...

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

    Science.gov (United States)

    Balasuriya, Udeni B R

    2014-01-01

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

  15. RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza

    DEFF Research Database (Denmark)

    Dybkaer, Karen; Munch, Mette; Handberg, Kurt Jensen; Jørgensen, Poul H

    A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected with...

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

    Science.gov (United States)

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

    2016-09-01

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

  17. Enteroviral pharyngitis diagnosed by reverse transcriptase-polymerase chain reaction.

    OpenAIRE

    Sharland, M.; Hodgson, J.; Davies, E G; Booth, J.; Jeffery, S

    1996-01-01

    The role of enteroviruses in childhood pharyngitis was investigated using enteroviral specific reverse transcriptase-polymerase chain reaction (RT-PCR). Viral/bacterial throat swabs were taken from 50 children with acute pharyngitis and 26 controls. A positive culture was identified in only 26% of children with pharyngitis (adenovirus 10%, group A streptococci 2%), and none of the controls. Enteroviral RT-PCR was positive in 8% of the pharyngitis group and none of the controls. Enteroviruses ...

  18. Deteksi Virus Classical Swine Fever di Bali dengan RT-PCR

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    I Wayan Wirata

    2010-09-01

    Full Text Available Classical Swine Fever (CSF virus has been confirmed for the first time in pig in Bali. The object of thisstudy was suspected CSF cases diagnosed at the diagnostic laboratory assistantship of the Faculty ofVeterinary Medicine, Udayana University, in 2007-2008. Total number of cases was 12. Case recordsincluded the signalment of case (breed, age, body weight, and the origin of respective case, clinical signs,post-mortem lesions, and histological pictures. CSF virus was confirmed using the standardized reversetranscriptase-polymerase chain reaction (RT-PCR for CSF from European Union. One RT-PCR productwas sequenced. CSF virus was confirmed in seven out of 12 cases (58%. The cDNA sequence wasconfirmed to be specific of CSF E2 protein coding region with 98% homology to one isolate from China thatwas available in GeneBank. Further works are recommended to elucidate the sensitivity of RT-PCR, toclarify some differential diagnose, and to find out the genetic variation of CSF virus in Bali.Key words: classical swine fever virus, Bali, RT-PCR

  19. Application and evaluation of RT-PCR-ELISA for the nucleoprotein and RT-PCR for detection of low-pathogenic H5 and H7 subtypes of avian influenza virus

    DEFF Research Database (Denmark)

    Dybkær, Karen; Munch, Mette; Handberg, Kurt J.; Jørgensen, Poul Henrik

    2004-01-01

    Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples...... originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT...

  20. Evaluation of a multiplex real-time polymerase chain reaction assay for the detection of influenza and respiratory syncytial viruses.

    Science.gov (United States)

    Esposito, Susanna; Scala, Alessia; Tagliabue, Claudia; Zampiero, Alberto; Bianchini, Sonia; Principi, Nicola

    2016-01-01

    Nasopharyngeal swabs from 424 children were used to compare the performances of the new multiplex real-time polymerase chain reaction (RT-PCR) RIDA®GENE Flu & RSV kit and monospecific RT-PCR assays in detecting respiratory syncytial and influenza viruses. The easy-to-use kit was highly sensitive and specific and is recommended for routine practice. PMID:26458277

  1. 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

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    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.

  2. Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

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    L.E. Alvares

    2003-12-01

    Full Text Available The reverse transcription-polymerase chain reaction (RT-PCR is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping ß-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19% was estimated for the different genes analyzed (6 to 9 repetitions. The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

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

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    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

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

  4. RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

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    Marinho Vera Lúcia A.

    2003-01-01

    Full Text Available A method to detect Apple stem grooving virus (ASGV based on reverse transcription polymerase chain reaction (RT-PCR was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV and Apple stem pitting virus (ASPV. The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

  5. Diagnosis of Dengue by Using Reverse Transcriptase-Polymerase Chain Reaction

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    Miagostovich Marize Pereira

    1997-01-01

    Full Text Available A rapid identification of dengue viruses from clinical samples by using a nested reverse transcriptase-polymerase chain reaction (RT-PCR procedure was carried out for diagnostic and epidemiological purposes. RT-PCR identified DEN-1 and DEN-2 viruses in 41% (41/100 of previously confirmed cases and provided an accurate confirmation of DHF in four fatal cases. RT-PCR was also useful for detecting and typing dengue viruses in suspected cases, allowing a rapid identification of new serotypes in endemic areas

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Willey James

    2004-01-01

    Full Text Available Abstract Background Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA in two cell lines (T24 & LD419 and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT in precisely standardized quantities along with the native template (NT in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as β-actin (ACTB. Results The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (t-test. StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p Conclusion StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further, it correlates well with Taqman real time PCR in terms of quantitative and discriminatory ability. This label-free, inexpensive technique may provide the ability to generate prognostically important molecular signatures unique to individual tumors and may enable identification of novel therapeutic targets.

  10. Detection of EML4-ALK in lung adenocarcinoma using pleural effusion with FISH, IHC, and RT-PCR methods.

    Directory of Open Access Journals (Sweden)

    Leilei Liu

    Full Text Available Anaplastic lymphoma kinase (ALK and echinoderm microtubule-associated protein-like 4 (EML4 gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC, leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the EML4-ALK fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The EML4-ALK fusion gene status was determined with fluorescent in situ hybridization (FISH, reverse transcription-polymerase chain reaction (RT-PCR, and immunohistochemistry (IHC. EML4-ALK was detected in three of 66 patient samples (4.5% with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the EML4-ALK fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting EML4-ALK in blocks of pleural effusion cells from lung cancer patients.

  11. Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing

    DEFF Research Database (Denmark)

    Wu, Jia Qian; Shteynberg, David; Arumugam, Manimozhiyan;

    2004-01-01

    alternative approach: reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on dual-genome de novo predictions from TWINSCAN. We tested 444 TWINSCAN-predicted rat genes that showed significant homology to known human genes implicated in disease but that were partially or...... single-intron experiment. Spliced sequences were amplified in 46 cases (34%). We conclude that this procedure for elucidating gene structures with native cDNA sequences is cost-effective and will become even more so as it is further optimized....

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

    Directory of Open Access Journals (Sweden)

    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

  13. 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...

  14. Targeting highly conserved 3'-untranslated region of pecluviruses for sensitive broad-spectrum detection and quantitation by RT-PCR and assessment of phylogenetic relationships.

    Science.gov (United States)

    Dieryck, B; Delfosse, P; Reddy, A S; Bragard, C

    2010-11-01

    The 3'-end region of many virus isolates has been shown to possess conserved sequences in addition to the presence of numerous genomic and subgenomic RNAs. Utilizing these sequences, a broad-spectrum reverse transcription-polymerase chain reaction protocol has been developed to detect all the known Indian peanut clump virus and Peanut clump virus isolates, that cause peanut clump diseases in West Africa and India. The primers were targeted at the highly conserved 3'-untranslated regions of the PCV RNA-1 and RNA-2. The conservation was confirmed by sequencing these untranslated regions of RNA-1 for six isolates and RNA-2 for one isolate. The conserved structure of the RNA-1 and RNA-2 was observed and the importance of this region for the virus survival was confirmed. The primers were also designed for virus quantitation using a Taqman(®)-based real-time RT-PCR. The use of RT-PCR and real-time quantitative RT-PCR improved the sensitivity of PCV detection compared to ELISA. RT-PCR also led to the detection of IPCV and PCV on two new natural hosts: Oldenlandia aspera and Vigna subterranea. Real-time RT-PCR is considered to be an ideal tool for identifying resistant sources to both IPCV and PCV. PMID:20723565

  15. Expression of sodium-lodide symporter (NlS) in thyroid nodules: comparison of RT-PCR and immunohistochemical staining methods

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Sang Kyun [College of Medicine, InJe Univ., Busan (Korea, Republic of); Lee, Kang Dae; Chang, Hee Kyung [College of Medicine, Kosin Univ., Busan (Korea, Republic of)

    2004-12-01

    The sodium-iodide symporter (NlS) expression is an important factor in determining the sensitivity of radioiodine therapy in well-differentiated thyroid cancers. Several previous studies for the expression of NlS in thyroid tissues show diverse results. To investigate whether there is difference between methods in determining the expression of NIS in thyroid tissues of patients with thyroid nodules, we measured the expression of NIS using two different methods (RT-PCR and immunohistochemical staining) and compared the results. We measured the expression of NIS by reverse transcriptase-polymerase chain reaction (RT-PCR) and also by immunohistochemical staining using anti-NIS antibody in thyroid cancers and other benign thyroid diseases. We compared the results of each method. We included 19 papillary carcinomas, 1 follicular carcinoma, 1 medullary carcinoma, 4 adenomas and 7 nodular hyperplasias. By RT-PCR analysis, 10 of 19 papillary carcinomas expressed NIS, but 1 follicular cancer didn't express NlS. By immunohistochemical staining, 15 of 19 papillary carcinomas express NlS, but 1 follicular cancer didn't express NIS. There was a significant correlation between the semiquantitative results of RT-PCR and immunohistochemical staining of NIS expression. (p<0.01) Our data demonstrated that the expression of NIS in thyroid cancers and other benign diseases investigated by RT-PCR and immunohistochemical staining correlated well each other. However, by immunohistochemical staining, more NIS expression was found.

  16. 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.

  17. Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

    OpenAIRE

    Marcos Lázaro Moreli; Ricardo Luiz Moro de Sousa; Luiz Tadeu Moraes Figueiredo

    2004-01-01

    We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positi...

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  1. A multiplex RT-PCR for simultaneous detection and identification of five viruses and two viroids infecting chrysanthemum.

    Science.gov (United States)

    Zhao, Xiting; Liu, Xingliang; Ge, Beibei; Li, Mingjun; Hong, Bo

    2015-05-01

    Pathogens causing significant economic losses in chrysanthemum include tomato aspermy virus (TAV), chrysanthemum virus B (CVB), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY), chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd). A multiplex reverse transcription polymerase chain reaction (RT-PCR) method, using specific primer sets for each virus or viroid, was developed for simultaneous detection and differentiation of TAV, CVB, CMV, TMV, PVY, CChMVd, and CSVd. The RT-PCR method was validated by testing chrysanthemum samples collected from different regions of China. In this study, CVB, TAV, TMV, PVY, CSVd, CMV, and CChMVd were detected, respectively, in 24.7 %, 17.5 %, 4.4 %, 4.4 %, 2.9 %, 2.5 %, and 1.5 % of the samples tested. These results indicate that CVB and TAV (24.7 % and 17.5 %) are common, whereas CMV, TMV, CChMVd, CSVd, and PVY (all below 5 %) are less frequently encountered. This new multiplex RT-PCR method has potential to be used routinely in large-scale virus and viroid surveys. PMID:25698104

  2. Rapid Detection Co-infections of Classical Swine Fever Virus and Porcine Reproductive and Respiratory Syndrome Virus by One-step Multiplex RT-PCR

    Institute of Scientific and Technical Information of China (English)

    TIAN Hong; WU Jinyan; YAN Chen; SHANG Youjun; YIN Shuanghui; LIU Xiangtao

    2011-01-01

    Classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) have caused immense economic loss in the pig industry and are considered to be the two most important infectious diseases of pigs in the world A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) was developed for CSFV and PRRSV co-infections or infections, respectively. A set of two pairs of primer was designed based on the sequence of nonstructural protein NS54B of CSFV and ORF7 gene of PRRSV. The diagnostic accuracy of multiplex RT-PCR assay was evaluated by using 56 field clinical samples by multiplex RT-PCR, single RT-PCR and sequence analysis; and the specificity of multiplex PCR was verified by using constructed plasmids containing the specific viral target fragments of PRRSV and CSFV, respectively. The results indicated that this assay could reliably differentiate PRRSV and CSFV in co-infection samples. The multiplex RT-PCR developed in this study might provide a new avenue to the rapid the detection of CSFV and PRRSV in one reaction.

  3. 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.

  4. 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. PMID:24509829

  5. Development of TaqMan real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus.

    Science.gov (United States)

    Zhu, Xiangdong; Wang, Yufei; Chen, Jianfei; Zhang, Xin; Shi, Hongyan; Shi, Da; Gao, Jing; Feng, Li

    2016-08-01

    Porcine kobuvirus (PKV) is a newly emerging virus that has been detected in diarrheic pigs. Presently, reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated amplification are the only methods that can be used to detect PKV. To develop a TaqMan real-time RT-PCR for the rapid detection and quantitation of PKV nucleic acid in fecal samples, a pair of primers and a probe were designed to amplify the conserved 3D region of the PKV genome. After optimization, the TaqMan real-time RT-PCR was highly specific and ∼1000 times more sensitive than conventional RT-PCR, and the detection limit was as low as 30 DNA copies. Among the 148 intestinal samples from piglets with diarrhea, 136 and 118 were positive based on the TaqMan and conventional RT-PCR methods, respectively, indicating that the TaqMan RT-PCR was more sensitive than conventional RT-PCR, and the total concordance of the two methods was approximately 87.84%. Thus, the TaqMan real-time RT-PCR should be a useful tool for the early detection and quantitation of PKV. PMID:26912233

  6. Chain reaction

    International Nuclear Information System (INIS)

    Chain Reaction is a work of recent American political history. It seeks to explain how and why America came to depend so heavily on its experts after World War II, how those experts translated that authority into political clout, and why that authority and political discretion declined in the 1970s. The author's research into the internal memoranda of the Atomic Energy Commission substantiates his argument in historical detail. It was not the ravages of American anti-intellectualism, as so many scholars have argued, that brought the experts back down to earth. Rather, their decline can be traced to the very roots of their success after World War II. The need to over-state anticipated results in order to garner public support, incessant professional and bureaucratic specialization, and the sheer proliferation of expertise pushed arcane and insulated debates between experts into public forums at the same time that a broad cross section of political participants found it easier to gain access to their own expertise. These tendencies ultimately undermined the political influence of all experts. (author)

  7. Webtag: a new web tool providing tags/anchors for RT-PCR experiments with prokaryotes

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2007-10-01

    Full Text Available Abstract Background Webtag is a tool providing oligonucleotide sequences (usually called tags or anchors that are absent from a specified genome. These tags/anchors can be appended to gene specific primers for reverse transcriptase polymerase chain reaction experiments, circumventing genomic DNA contamination. Results The use of a relational database, in conjunction with a series of scripts written in PHP and Perl, allows the user to rapidly obtain tags that are: 1 suitable for a specific organism, and 2 compatible with other oligonucleotides to be used in the experimental procedures. Conclusion This new web tool allows scientists to easily and rapidly obtain suitable tags for RT-PCR experiments, and is available at http://www.egs.uu.se/software/webtag/.

  8. 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.

  9. Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

    Directory of Open Access Journals (Sweden)

    Anny Armas Cayarga

    2011-01-01

    Full Text Available Human immunodeficiency virus type-1 (HIV-1 viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (=0.925 was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux for HIV-1 RNA quantitation with clinical samples (=14. HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load.

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

    Directory of Open Access Journals (Sweden)

    Karl Kashofer

    Full Text Available 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

  11. Simultaneous Detection of Three Arboviruses Using a Triplex RT-PCR Enzyme Hybridization Assay

    Institute of Scientific and Technical Information of China (English)

    Dan Dong; Shi-hong Fu; Li-hua Wang; Zhi Lv; Tai-yuan Li; Guo-dong Liang

    2012-01-01

    Arboviruses represent a serious problem to public health and agriculture worldwide.Fast,accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation.We developed a cost-effective,rapid,and highly sensitive one-step "triplex RT-PCR enzyme hybridization"assay for simultaneous detections of Japanese Encephallitis virus (JEV,Flaviviridae)Getah virus (GETV,Togaviridae),and Tahyna virus (TAHV,Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction.The analytical sensitivity of this assay was 1 PFU/mL for JEV,10PFU/mL for GETV,and 10 PFU/mL for TAHV.This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods.When “triplex RT-PCR enzyme hybridization” was applied to 29 cerebrospinal fluid(CSF)samples that were JEV-positive by normal RT-PCR assay,all samples were strongly positive for JEV,but negative for GETV and TAHV,demonstrating a good sensitivity,specificity,and performance at CSF specimen detection.

  12. 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...... by the generic rRT-PCR diagnostic assays but negative by virus isolation and antigen-detection ELISA which would otherwise have to be serotyped by nucleotide sequencing. A similar approach could be used to develop serotyping assays for FMDV strains circulating in other regions of the world....

  13. Diagnosis of Infectious Bronchitis Disease in Broiler Chickens by Serological Test (ELISA and RT-PCR in Duhok

    Directory of Open Access Journals (Sweden)

    Dyar Adil Morad AL-Barwary

    2012-02-01

    Full Text Available Infectious Bronchitis (IB is one of the most important viral diseases of poultry and it causes major economic losses in poultry industry. The study was conducted to detect Infectious Bronchitis virus (IBV in broilers chicken farms in Duhok Governorate. To achieve this goal two tests have been used throughout the study protocol Enzyme-Linked Immunosorbent Assay (ELISA and Reverse Transcription Polymerase Chain Reaction (RT-PCR. One hundred and eighty Serum samples were collected from 11 chicken farms including (120 samples from 9 symptomatic non vaccinated chicken farms, (30 samples from one asymptomatic vaccinated chicken farm and another (30 samples from one asymptomatic non vaccinated chicken farm and screened for the presence of IBV antibodies by ELISA kit. The total RNAs were extracted from tracheal tissues using RNX TM-Plus reagent (Cinnagen,Iran. Eighty serum samples were positive: 50/120 (41.6% sample were symptomatic non vaccinated, another 30/30 (100% were asymptomatic vaccinated chickens samples, and the remaining samples (100 were negative by ELISA , including 70 /120 (58.4% from symptomatic non vaccinated chickens, and 30/30 (100% from asymptomatic non vaccinated chickens. The molecular detection of avian infectious bronchitis virus by use of RT-PCR was applied to extract RNAs from tracheal tissues. The test was performed on (80 symptomatic non vaccinated chickens and 36 vaccinated and non vaccinated chickens. Detection was performed using universal types of primers including XCE2+ and XCE2- 80 symptomatic chickens were examined by RT-PCR (50 with positive ELISA and 30 with negative ELISA results, 36 (44.4 % have detectable IBV-cDNA and the remaining 44 (55.6% did not have detectable IBVcDNA. Another 36 (18 asymptomatic vaccinated and 18 non vaccinated chickens were examined by RT-PCR, 8 (22.2% have detectable IBV-cDNA, and the remaining 28 (77.8% were negative. This study has determined the presences of IBV in flocks of Duhok

  14. Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

    Science.gov (United States)

    Brault, Aaron C; Fang, Ying; Reisen, William K

    2015-05-01

    Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk. PMID:26334826

  15. THE APLICATION OF REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION FOR THE DIAGNOSIS OF CANINE DISTEMPER

    OpenAIRE

    I Nyoman Suartha; I Gusti Ngurah Kade Mahardika; Ida Ayu Sri Candra Dewi; Ni Ketut Dias Nursanty; Yosaphat L.S Kote; Anita Dwi Handayani; I Gusti Agung Ayu Suartini

    2008-01-01

    A study was conducted to apply reverse transcriptase-polymerase chain reaction (RT-PCR) technique for the confirmative diagnosis of canine distemper in dogs. Twenty mongreal dogs with clinical symptoms of canine distemper were used in this study. The viral RNA was isolated from nasal swab using Trizol® and transcribed into cDNA using random primers 5’ACAGGATTGCTGAGGACCTAT 3’. The cDNA was amplified in one step RT-PCR using primers 5’-ACAGGATTGCTGAGGACCTAT-3’ (forward) and 5’- CAAGATAACCATGTAC...

  16. 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.

  17. Detection and Comparison of Pathogen of Virus Disease in Pumpkin by RT-PCR and IC-PCR

    Institute of Scientific and Technical Information of China (English)

    YANG Guohui; ZHANG Zhongkai; CUI Chongshi

    2006-01-01

    Two kinds of methods RT-PCR and IC-PCR were used to detect pathogen of virus disease of pumpkin and the sensitivity of the two methods was compared. The results showed that PRSV-W and CMV were detected in diseased samples gathered in Yunnan Province, while WMV and CMV were detected in diseased samples gathered in Heilongjiang Province. The sensitivity of RT-PCR is higher than that of IC-PCR, but the effect of IC-PCR in the specialization of bonding reaction and requisition for experiment material is better than that of RT-PCR.

  18. THE APLICATION OF REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION FOR THE DIAGNOSIS OF CANINE DISTEMPER

    Directory of Open Access Journals (Sweden)

    I Nyoman Suartha

    2008-03-01

    Full Text Available A study was conducted to apply reverse transcriptase-polymerase chain reaction (RT-PCR technique for the confirmative diagnosis of canine distemper in dogs. Twenty mongreal dogs with clinical symptoms of canine distemper were used in this study. The viral RNA was isolated from nasal swab using Trizol® and transcribed into cDNA using random primers 5’ACAGGATTGCTGAGGACCTAT 3’. The cDNA was amplified in one step RT-PCR using primers 5’-ACAGGATTGCTGAGGACCTAT-3’ (forward and 5’- CAAGATAACCATGTACGGTGC-3’ (backward. A single band of 300 bp which was specific for canine distemper virus CDV was detected in fifteen out of twenty samples. It is therefore evident that confirmative diagnostics of canine distemper disease can be established with RT-PCR technique.

  19. Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Screening of 29 Chromosomal Translocation in Hematologic Malignancies

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Multiplex reverse transcription-polymerase chain reaction (M-RT-PCR) has been proved to possess great clinical potential for simultaneous screening of 29 chromosomal translocations in acute leukemia. To evaluate the clinical value of M-RT-PCR in hematologic malignancies, bone marrow samples from 90 patients with various hematologic malignancies, including 25 acute myeloge nous leukemia (AML), 22 acute lymphoblastic leukemia (ALL), 27 chronic myelogenous leukemia (CML), 4 myeloproliferative diseases (MPD), 3 chronic lymphoblastic leukemia (CLL), 3 and 1 malignant histocytosis (MH) were subjected to both M-RT-PCR and chromosome karyotypic analysis. Some of cases were subjected to follow-up examination of M-RT-PCR during the period of ukemia. In our hand, 12 of 29chromosomal translocation transcripts including TEL/PDGFR, DEK/CAN, MLL/AF6, AML1/ETO,F9, BCR/ABL, MLL/MLL, PML/RARα, TLS/ERG, E2A/HLF, EVIl and HOXI1 were detected in 57 cases (63.3 %) of the 90 samples, which were in consistence with the results of karyore, M-RT-PCR had also shown good clinical relevance when used as an approach to detect minimal residual leukemia. We concluded that M-RT-PCR could be used as an effiy in the initial diagnosis of hematologic malignancies but also in subsequent monitor of minimal residual leukemia.

  20. Diagnosis of foot-and mouth disease by real time reverse transcription polymerase chain reaction under field conditions in Brazil

    Directory of Open Access Journals (Sweden)

    Clarke Neville P

    2008-12-01

    Full Text Available Abstract Background Foot-and-mouth disease (FMD is an economically important and highly contagious viral disease that affects cloven-hoofed domestic and wild animals. Virus isolation and enzyme-linked immunosorbent assay (ELISA are the gold standard tests for diagnosis of FMD. As these methods are time consuming, assays based on viral nucleic acid amplification have been developed. Results A previously described real-time reverse transcriptase polymerase chain reaction (RT-PCR assay with high sensitivity and specificity under laboratorial and experimental conditions was used in the current study. To verify the applicability of this assay under field conditions in Brazil, 460 oral swabs from cattle were collected in areas free of FMD (n = 200 and from areas with outbreaks of FMD (n = 260. Three samples from areas with outbreaks of FMD were positive by real-time RT-PCR, and 2 of those samples were positive by virus isolation and ELISA. Four other samples were considered inconclusive by real-time RT-PCR (threshold cycle [Ct] > 40; whereas all 200 samples from an area free of FMD were real-time RT-PCR negative. Conclusion real-time RT-PCR is a powerful technique for reliable detection of FMDV in a fraction of the time required for virus isolation and ELISA. However, it is noteworthy that lack of infrastructure in certain areas with high risk of FMD may be a limiting factor for using real-time RT-PCR as a routine diagnostic tool.

  1. Penerapan Metode Diagnosis Cepat Virus Avian Influenza H5N1 dengan Metode Single Step Multiplex RT-PCR

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2010-12-01

    Full Text Available Avian influenza (AI virus is a segmented single stranded (ss RNA virus with negative polarity andbelong to the Orthomyxoviridae family. Diagnose of AI virus can be performed using conventional methodsbut it has low sensitivity and specificity. The objective of the research was to apply rapid, precise, andaccurate diagnostic method for AI virus and also to determine its type and subtype based on the SingleStep Multiplex Reverse Transcriptase-Polymerase Chain Reaction targeting M, H5, and N1 genes. In thismethod M, H5 and NI genes were simultaneously amplified in one PCR tube. The steps of this researchconsist of collecting viral RNAs from 10 different AI samples originated from Maros Disease InvestigationCenter during 2007. DNA Amplification was conducted by Simplex RT-PCR using M primer set. Then, bysingle step multiplex RT-PCR were conducted simultaneously using M, H5 and N1 primers set. The RTPCRproducts were then separated on 1.5% agarose gel, stained by ethidum bromide and visualized underUV transilluminator. Results showed that 8 of 10 RNA virus samples could be amplified by Simplex RTPCRfor M gene which generating a DNA fragment of 276 bp. Amplification using multiplex RT-PCRmethod showed two of 10 samples were AI positive using multiplex RT-PCR, three DNA fragments weregenerated consisting of 276 bp for M gene, 189 bp for H5 gene, and 131 bp for N1. In this study, rapid andeffective diagnosis method for AI virus can be conducted by using simultaneous Single Step Multiplex RTPCR.By this technique type and subtype of AI virus, can also be determined, especially H5N1.

  2. Development of an in situ magnetic beads based RT-PCR method for electrochemiluminescent detection of rotavirus

    Science.gov (United States)

    Zhan, Fangfang; Zhou, Xiaoming

    2012-12-01

    Rotaviruses are double-stranded RNA viruses belonging to the family of enteric pathogens. It is a major cause of diarrhoeal disease in infants and young children worldwide. Consequently, rapid and accurate detection of rotaviruses is of great importance in controlling and preventing food- and waterborne diseases and outbreaks. Reverse transcription-polymerase chain reaction (RT-PCR) is a reliable method that possesses high specificity and sensitivity. It has been widely used to detection of viruses. Electrochemiluminescence (ECL) can be considered as an important and powerful tool in analytical and clinical application with high sensitivity, excellent specificity, and low cost. Here we have developed a method for the detection of rotavirus by combining in situ magnetic beads (MBs) based RT-PCR with ECL. RT of rotavirus RNA was carried out in a traditional way and the resulting cDNA was directly amplified on MBs. Forward primers were covalently bounded to MBs and reverse primers were labeled with tris-(2, 2'-bipyridyl) ruthenium (TBR). During the PCR cycling, the TBR labeled products were directly loaded and enriched on the surface of MBs. Then the MBs-TBR complexes could be analyzed by a magnetic ECL platform without any post-modification or post-incubation which avoid some laborious manual operations and achieve rapid yet sensitive detection. In this study, rotavirus from fecal specimens was successfully detected within 2 h, and the limit of detection was estimated to be 104copies/μL. This novel in situ MBs based RT-PCR with ECL detection method can be used for pathogen detection in food safety field and clinical diagnosis.

  3. Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids

    Directory of Open Access Journals (Sweden)

    Fábio Takenori Higa

    2013-04-01

    Full Text Available We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.

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

    Science.gov (United States)

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

    2014-11-01

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

  5. Detection and Molecular Characterization of Enteroviruses in Korean Surface Water by Using Integrated Cell Culture Multiplex RT-PCR

    Institute of Scientific and Technical Information of China (English)

    GYUCHEOL LEE; CHANHEE LEE; CHANSEUNG PARK; SANGGI JEONG

    2008-01-01

    Objective To identify waterborne enteric viruses in Korean surface water. Methods Integrated cell culture(ICC)-multiplex reverse transcription-polymerase chain reaction (RT-PCR) was simultaneously designed to detect coxsackieviruses (CV), polioviruses (PV), and reoviruses (RV). ICC-multiplex RT-PCR and phylogenetic analysis were conducted using 21 total culturable virus assay (TCVA)-positive sample-inoculated cell cultures. Results CV and RV were detected in 9 samples each, and 3 samples were positive for both CV and RV. PV was not etected in any sample. Molecular phylogenetic analysis of the VPl gene sequences revealed that CV types B2 and B4 redominated in Korean surface water, and the nucleotide sequences of CV type B2 were clustered with those of CVs isolated from China and Japan. The results suggested that the evolution of these viruses occurred in a region-specific manner. Conclusion CV and RV are detectable in Korean surface water, with a predominance of CV type B2, and the evolution of CV type B2 occur in a region-specific manner.

  6. Impact of coccidial infection on vaccine- and vvIBDV in lymphoid tissues of SPF chickens as detected by RT-PCR

    Directory of Open Access Journals (Sweden)

    Bisgaard Magne

    2006-09-01

    Full Text Available Abstract Background This study aimed at investigating a potential effect caused by coccidia on the immune response to vaccine- and very virulent infectious bursal disase virus (vvIBDV in SPF chickens. Methods Two groups of three weeks old SPF chickens were vaccinated prior to inoculation with coccidia and challenge with virulent IBDV, all within a period of eight days. Two control groups were similarly treated, except that challenge with field virus was omitted in one group while inoculation with coccidia was omitted in the other group. Clinical signs, lesions in the intestines caused by coccidia, lesions in the bursa of Fabricius caused by IBDV, IBDV-antibody titres, and virus detection by reverse transcription polymerase chain reaction (RT-PCR were compared among the groups. Lymphoid tissues and swab samples were analysed by general RT-PCR, and positive results were identified by strain specific duplex (DPX RT-PCR. Results In the tripple-infected groups, vaccine strain IBDV was detected in spleen and thymus tissues, and no field virus was detected in bursa samples, contrary to the double-infected groups. Conclusion The results suggest an enhancing effect on the immune response caused by subclinical coccidiosis and vvIBDV acting in concert.

  7. Development of pan-phlebovirus RT-PCR assay.

    Science.gov (United States)

    Klimentov, Alexander S; Butenko, Alexander M; Khutoretskaya, Natalia V; Shustova, Elena Yu; Larichev, Victor F; Isaeva, Olga V; Karganova, Galina G; Lukashev, Alexander N; Gmyl, Anatoly P

    2016-06-01

    This study reports the pan-phlebovirus assay capable of detecting both sandfly/mosquito- and tick-borne phleboviruses. Sensitivity and specificity of the assay was verified using a panel of arboviruses. The RT-PCR assay is simple and sensitive, and thus well suited for screening of field samples. PMID:26947398

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

    Institute of Scientific and Technical Information of China (English)

    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.

  9. Real-time polymerase chain reaction for diagnosis and quantitation of negative strand of chikungunya virus.

    Science.gov (United States)

    Chiam, Chun Wei; Chan, Yoke Fun; Loong, Shih Keng; Yong, Sara Su Jin; Hooi, Poh Sim; Sam, I-Ching

    2013-10-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is useful for diagnosis and studying virus replication. We developed positive- and negative-strand qRT-PCR assays to detect nsP3 of chikungunya virus (CHIKV), a positive-strand RNA alphavirus that causes epidemic fever, rash, and arthritis. The positive- and negative-strand qRT-PCR assays had limits of quantification of 1 and 3 log10 RNA copies/reaction, respectively. Compared to a published E1 diagnostic assay using 30 laboratory-confirmed clinical samples, the positive-strand nsP3 qRT-PCR assay had higher R(2) and efficiency and detected more positive samples. Peak viral load of 12.9 log(10) RNA copies/mL was reached on day 2 of illness, and RNA was detectable up to day 9, even in the presence of anti-CHIKV IgM. There was no correlation between viral load and persistent arthralgia. The positive-strand nsP3 assay is suitable for diagnosis, while the negative-strand nsP3 assay, which uses tagged primers to increase specificity, is useful for study of active viral replication kinetics. PMID:23886793

  10. Survey and RT-PCR Based Detection of Cardamom mosaic virus Affecting Small Cardamom in India.

    Science.gov (United States)

    Biju, C N; Siljo, A; Bhat, A I

    2010-10-01

    Mosaic or marble or katte disease caused by Cardamom mosaic virus (CdMV) is an important production constraint in all cardamom growing regions of the world. In the present study, 84 cardamom plantations in 44 locations of Karnataka and Kerala were surveyed. The incidence of the disease ranged from 0 to 85%. The incidence was highest in Madikeri (Karnataka) while no incidence was recorded in Peermade (Kerala). In general, incidence and severity of the disease was higher in cardamom plantations of Karnataka. A procedure for total RNA isolation from cardamom and detection of CdMV through reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting the conserved region of coat protein was standardized and subsequently validated by testing more than 50 field cardamom samples originating from Karnataka and Kerala states. The method can be used for indexing the planting material and identifying resistant lines/cultivars before either they are further multiplied in large scale or incorporated in breeding. PMID:23637495

  11. Multiplex Reverse Transcription-Polymerase Chain Reaction untuk Deteksi Cepat Virus Flu Burung H5N1 (MULTIPLEX REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION FOR RAPID DETECTION OF H5N1 AVIAN INFLUENZA VIRUS

    Directory of Open Access Journals (Sweden)

    Raden Wasito

    2015-05-01

    Full Text Available Avian influenza virus subtype H5N1 (AIV H5N1 is highly pathogenic and fatal in poultry. The virusis still endemic with low virulence rate, although it may play a critical role in causing high morbidity andmortality rates in poultry in Indonesia. In general, diagnostic approach for AIV H5N1 is based onconventional serological and viral isolation methods that have the potential to produce consumings oftime and relatively expensive cost within the laboratory without compromising test utility. Thus, amolecular approach of multiplex reverse transcription-polymerase chain reaction (mRT-PCR was developedand applied for the detection of matrix gene type A influenza viruses, AIV subtype subtype H5hemagglutinin gene with simultaneous detection of N1 nucleoprotein gene. Thirty sera specimens fromthe diseased commercial chickens that were specifically amplified positive-RT-PCR for AIV H5N1 wereselected for mRT-PCR. The mRT-PCR products were visualized by agarose gel electrophoresis and consistedof DNA fragments of AIV of 245 bp, 545 bp and 343 bp for M, H5 and N1 genes, respectively. Thus, themRT-PCR that can rapidly differentiate simultaneously between these genes is very important for thecontrol and even eradication of AIV transmission in poultry in Indonesia.

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

    OpenAIRE

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

    2006-01-01

    International audience Foot-and-mouth disease virus (FMDV) can be excreted in milk and thereby spread infection to susceptible animals in other holdings. The feasibility of using real-time reverse transcription polymerase chain reaction (rRT-PCR) as a diagnostic tool for detection of FMDV in milk was assessed by studying the excretion of virus from experimentally-infected cattle. Fore- and machine milk samples were collected over a 4-week period from two dairy cows infected with FMDV and f...

  13. Comparison of real-time and quantitative polymerase chain reaction assays in detection of cytomegalovirus DNA in clinical specimens

    International Nuclear Information System (INIS)

    To compare the real-time (RT) and qualitative (Q) polymerase chain reaction (PCR) assays for detection of Cytomegalovirus (CMV) DNA. The study took place in the Department of Microbiology, Erciyes University, Kayseri and in Iontek Laboratory, Istanbul, Turkey, from August to December 2006. One hundred and seven clinical specimens from 67 patients were included in the study. Cytomegalovirus DNA was investigated using RT-PCR kit (Fluorion Iontek, Turkey) and Q-PCR kit (Fluorion Iontek, Turkey). Deoxyribonucleic acid sequencing was applied to the samples that yielded discrepant results in both assays. Mac Nema's Chi Square test was used for statistical analysis. Of the specimens, 27 were found positive with both assays: 9 with only RT-PCR, and 11 with only Q-PCR assay. Both assays were found negative in 60 of the specimens. There was a good agreement between the 2 assays in 87(81.3%) of the specimens. There was no statistical significant difference between the assays (p>0.05). Two of the 11 samples that RT-PCR negative Q-PCR positive, and 3 of 9 samples that RT-PCR positive Q-PCR negative were found to be CMV DNA positive by DNA sequencing. A good level of concordance between RT-PCR and Q-PCR assays for CMV DNA detection has been found. (author)

  14. A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health

    OpenAIRE

    Hoffmann, Bernd; Beer, Martin; Reid, Scott M.; Mertens, Peter,; Oura, Chris A.L.; van Rijn, Piet A.; Slomka, Marek J.; Banks, Jill; Brown, Ian H.; Alexander, Dennis J.; King, Donald P.

    2009-01-01

    Abstract Real-time, reverse transcription polymerase chain reaction (rRT-PCR) has become one of the most widely used methods in the field of molecular diagnostics and research. The potential of this format to provide sensitive, specific and swift detection and quantification of viral RNAs has made it an indispensable tool for state-of-the-art diagnostics of important human and animal viral pathogens. Integration of these assays into automated liquid handling platforms for nucleic a...

  15. Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

    Science.gov (United States)

    Modi, Arpan; Kumar, Nitish; Narayanan, Subhash

    2016-01-01

    Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4. PMID:27108325

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

    Science.gov (United States)

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

  17. Avaliação da urina e de leucócitos como amostras biológicas para a detecção ante mortem do vírus da cinomose canina por RT-PCR em cães naturalmente infectados Evaluation of the urine and leucocytes as biological samples for ante mortem detection of canine distemper virus by RT-PCR assay in naturally infected dogs

    OpenAIRE

    F.J. Negrão; A.A. Alfieri; A.F. Alfieri

    2007-01-01

    Urine and leucocytes were comparatively evaluated as clinical samples for ante mortem detection of the canine distemper virus (CDV) by a reverse transcription-polymerase chain reaction (RT-PCR) assay. One hundred and eighty eight dogs with clinical symptoms of distemper, were distributed in three groups. The group A was constituted of 93 dogs with systemic signs of distemper; the group B by 11 dogs with neurological signs, and the group C by 84 dogs that presented simultaneously systemic and ...

  18. Polymerase chain reaction-based discrimination of viable from non-viable Mycoplasma gallisepticum

    Directory of Open Access Journals (Sweden)

    Ching Giap Tan

    2014-02-01

    Full Text Available The present study was based on the reverse transcription polymerase chain reaction (RT-PCR of the 16S ribosomal nucleic acid (rRNA of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20–25 h at 37 °C, 22–25 h at 16 °C, and 23–27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h. The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.

  19. Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

    International Nuclear Information System (INIS)

    Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation

  20. RT-PCR standardization and bone mineralization after low-level laser therapy on adult osteoblast cells

    Science.gov (United States)

    do Bomfim, Fernando R. C.; Sella, Valéria R. G.; Zanaga, Jéssica Q.; Pereira, Nayara S.; Nouailhetas, Viviane L. A.; Plapler, Hélio

    2014-03-01

    Purpose: Osteoblasts are capable to produce different compounds directly connected to bone mineralization process. This study aims to standardize the reverse transcriptase polymerase chain reaction (RT-PCR) for adult osteoblasts to observe the effect of low level laser therapy on bone mineralization. Methods: Five-millimeter long fragments obtained from the mead femoral region of male Wistar rats were assigned into group A (n=10, laser) and group B (n=10, no laser), submitted to mechanic and enzymatic digestion. After 7 days, cultures of group A were irradiated daily on a single spot with a GaInAs laser, λ=808nm, 200mW/cm2, 2J/cm2, bean diameter of 0,02mm, 5 seconds for 6 days. Group B was manipulated but received no laser irradiation. After 13 days the cells were trypsinized for 15 minute and stabilized with RNA later® for RNA extraction with Trizol®. cDNA synthesis used 10μg of RNA and M-MLV® enzyme. PCR was accomplished using the β-actin gene as a control. Another aliquot was fixed for Hematoxylin-Eosin and Von Kossa staining to visualize bone mineralization areas. Results: Under UV light we observed clearly the amplification of β-actin gene around 400bp. HE and Von Kossa staining showed osteoblast clusters, a higher number of bone cells and well defined mineralization areas in group A. Conclusion: The cell culture, RNA extraction and RT-PCR method for adult osteoblasts was effective, allowing to use these methods for bone mineralization studies. Laser improved bone mineralization and further studies are needed involving osteogenesis, calcium release mechanisms and calcium related channels.

  1. Detection and strain differentiation of infectious bronchitis virus in tracheal tissues from experimentally infected chickens by reverse transcription-polymerase chain reaction. Comparison with an immunohistochemical technique

    DEFF Research Database (Denmark)

    Handberg, Kurt; Nielsen, O.L.; Pedersen, M.W.; Jørgensen, Poul Henrik

    1999-01-01

    Oligonucleotide pairs were constructed for priming the amplification of fragments of nucleocapsid (N) protein and spike glycoprotein (S) genes of avian infectious bronchitis virus (IBV) by reverse transcription-polymerase chain reaction (RT-PCR). One oligonucleotide pair amplified a common segment...

  2. Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting Tomato using 18S rRNA as an internal control

    Institute of Scientific and Technical Information of China (English)

    Shaoning Chen; Hao Gu; Xiaoming Wang; Jishuang Chen; Weimin Zhu

    2011-01-01

    A multiplex reverse-transcription polymerase chain reaction (RT-PCR) protocol was developed for simultaneous detection and discrimination of subgroups of Cucumber mosaic virus (CMV), including its satellite RNA, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV),using 18S rRNA as an internal control.Species- and subgroups-specific primers designed to differentiate CMV subgroups Ⅰ and Ⅱ, ToMV and TMV, were assessed using the cDNA clones of viral genomes, CMV satellite RNA and 18S rRNA gene from tomato (Solanum lycopersicum L.) or tobacco (Nicotiana tobacum).Using total RNA extracted from artificial mixture of tomato leaf tissues infected by each virus, the reaction components and cycling parmeters were optimized and a multiplex RT-PCR procedure was established.Six fragments of 704, 593, 512, 421,385, 255 bp, specific to CMV subgroup ll, CMV subgroup I, ToMV, TMV, satellite RNA and 18S rRNA, respectively, were sinultaneously amplified.The sensitivity of the multiplex RT-PCR method for detecting CMV was 100 times higher than that of double-antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA).This method was successfully used for field detection.Among 141 samples collected from East China through tomato growth seasons, 106 single infections with one of the above isolates were detected and 13 mixed infections were found.The results showed the potential use of this method for investigating the epidemiology of viral diseases infecting tomato.

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

    Science.gov (United States)

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

  4. Chain Reaction Polymerization.

    Science.gov (United States)

    McGrath, James E.

    1981-01-01

    The salient features and importance of chain-reaction polymerization are discussed, including such topics as the thermodynamics of polymerization, free-radical polymerization kinetics, radical polymerization processes, copolymers, and free-radical chain, anionic, cationic, coordination, and ring-opening polymerizations. (JN)

  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. Diagnosis of nervous necrosis virus in orange-spotted grouper, Epinephelus coioides, by a rapid and convenient RT-PCR method

    Institute of Scientific and Technical Information of China (English)

    MU Yinnan; LIN Kebing; CHEN Xinhua; AO Jingqun

    2013-01-01

    Viral nervous necrosis (VNN) causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction (RT-PCR) was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necro-sis virus (NNV ), in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus (IPNV ), infectious hematopoietic necrosis virus (IHNV ), spring viraemia of carp virus (SVCV ), epizootic haematopoietic necrosis virus (EHNV ), and large yellow croaker iridovirus (LYCIV ). With this method, the orange-spotted grouper (Epinephelus coioides) fry from hatcheries with or without incidence of the VN-N epidemic in Fujian Province were detected. The results showed that all or 93%of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40%or 25%of fry from the t-wo hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.

  7. Detection of potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR.

    Science.gov (United States)

    Arif, Muhammad; Ali, Murad; Rehman, Anayatur; Fahim, Muhammad

    2014-01-01

    The hilly region of Northwest of Pakistan is leading seed potato producing areas of the country. Soil and plant samples were collected from the region and tested for PMTV using both conventional and molecular techniques. The bait plants exhibited PMTV-characteristic v-shaped yellow leaf markings in Nicotiana debneyi plants grown in putative viruliferious soils from 20/26 locations. The results were confirmed by back inoculation of sap from both roots and leaves of bait plant on indicator hosts (N. debneyi, Nicotiana benthamiana). The root samples of bait plants grown in soils of 25 locations and leaves of 24 locations reproduced systemic infection on indicator hosts upon back inoculation. The virus was identified in bait plants grown in soils from 25/26 locations using double antibody sandwich-enzyme linked immunosorbent assay (DAS)-ELISA and reverse transcription and polymerase chain reaction (RT-PCR) methods. The products of the 566bp were amplified from coat protein region of PMTV RNA 3 in both root and leaf samples of baited plants. The virus was detected in 10 potato cultivars commercially grown in the region using DAS-ELISA and RT-PCR. The virus was also detected in zoospores of Spongospora subterranea derived from the peels of selected scabby tubers using triple antibody sandwich (TAS)-ELISA. The results indicate that a bait plant bioassay, infectivity assay, ELISA and RT-PCR can detect PMTV in roots and leaves of baited plants, field samples, zoospores of S. subterranea and tubers of 10 potato cultivars commercially grown in the region. PMID:24161813

  8. Typing of Poultry Influenza Virus (H5 and H7 by Reverse Transcription- Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Cesare Bonacina

    2010-01-01

    Full Text Available The ability of the influenza Orthomixovirus to undergo to continually antigenically changes that can affect its pathogenicity and its diffusion, explains the growing seriousness of this disease and the recent epizoozies in various parts of the world. There have been 15 HA and 9 NA type A sub-types of the influenza virus identified all of which are present in birds. Until now the very virulent avian influenza viruses identified were all included to the H5 and H7 sub-types. We here show that is possible to identify the H5 and H7 sub-types with reverse transcription-polymerase chain reaction (RT-PCR by using a set of specific primers for each HA sub-type. The RT-PCR is a quick and sensitive method of identifying the HA sub-types of the influenza virus directly from homogenised organs.

  9. Survey of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus incidence in Korea by Duplex RT-PCR

    Directory of Open Access Journals (Sweden)

    Seung-Yeol Lee

    2014-12-01

    Full Text Available The incidence of Cherry necrotic rusty mottle virus (CNRMV and Cherry green ring mottle virus (CGRMV have recently been occurred in Korea, posing a problem for sweet cherry cultivation. Since infected trees have symptomless leaves or ring-like spots on the pericarp, it is difficult to identify a viral infection. In this study, the incidence of CNRMV and CGRMV in sweet cherry in Gyeongbuk province was surveyed using a newly developed duplex reverse transcriptase polymerase chain reaction (RT-PCR method that can detect both viruses in a single reaction. CNRMV and CGRMV co-infection rates were 29.6%, 53.6%, and 17.6%, respectively, in samples collected from three different sites (Daegu, Gyeongju and Gyeongsan in Gyeongbuk province during 2012 and 2013. This duplex RT-PCR method offers a simple, rapid, and effective way of identifying CNRMV and CGRMV simultaneously in sweet cherry trees, which can aid in the management of viral infections that could undermine yield.

  10. Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Dong, X Y; Li, W H; Zhu, J L; Liu, W J; Zhao, M Q; Luo, Y W; Chen, J D

    2015-01-01

    Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance. PMID:27175171

  11. Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

    Directory of Open Access Journals (Sweden)

    Paschoalin Vânia MF

    2005-04-01

    Full Text Available Abstract Background Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target. Results We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65°C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I or, alternatively, they were directly used in RT-PCR reactions (protocol II. Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step. Conclusion Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.

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

    Science.gov (United States)

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

    2016-01-01

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

  13. A multiple RT-PCR assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees.

    Science.gov (United States)

    Hao, Lu; Xie, Jipeng; Chen, Shanyi; Wang, Shaojie; Gong, Zhuoqun; Ling, Kai-Shu; Guo, Liyun; Fan, Zaifeng; Zhou, Tao

    2016-08-01

    Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-PCR (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1α (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs. PMID:27054889

  14. Are PEI-coated SWCNTs conjugated with hepatitis A virus? A chemical study with SEM, Z-potential, EDXD and RT-PCR

    International Nuclear Information System (INIS)

    The conjugation between nanotubes, coated with different doses of polyethylene imine (PEI) and hepatitis A virus (HAV) was investigated by scanning electron microscopy, Z-potential, thermogravimetric and differential thermal analysis, transmission electron microscopy, energy dispersive x-ray diffraction (EDXD) and reverse transcript polymerase chain reaction (RT-PCR). For the first time, to our knowledge, evidence is obtained that conjugation between the nanotubes and the HAV occurs and that it has an (at least a partial) electrostatic character. Since all components of the conjugated systems, nanotubes, coating material and virus are characterized by different peak shapes in the selected q range, it was possible to infer that conjugation occurred. RT-PCR measurements confirmed that the conjugation of the coated nanotubes and HAV occurred and the result was stable. This opens up the prospect of probing the coated nanotubes as intra-cellular carriers in transfection processes of the virus. Further biological applications will concern a possible vaccine especially for non-replicative viruses.

  15. Selection and Verification of Candidate Reference Genes for Mature MicroRNA Expression by Quantitative RT-PCR in the Tea Plant (Camellia sinensis).

    Science.gov (United States)

    Song, Hui; Zhang, Xiao; Shi, Cong; Wang, Shuangshuang; Wu, Ailin; Wei, Chaoling

    2016-01-01

    Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a rapid and sensitive method for analyzing microRNA (miRNA) expression. However, accurate qRT-PCR results depend on the selection of reliable reference genes as internal positive controls. To date, few studies have identified reliable reference genes for differential expression analysis of miRNAs among tissues, and among experimental conditions in plants. In this study, three miRNAs and four non-coding small RNAs (ncRNA) were selected as reference candidates, and the stability of their expression was evaluated among different tissues and under different experimental conditions in the tea plant (Camellia sinensis) using the geNorm and NormFinder programs. It was shown that miR159a was the best single reference gene in the bud to the fifth leaf, 5S rRNA was the most suitable gene in different organs, miR6149 was the most stable gene when the leaves were attacked by Ectropis oblique and U4, miR5368n and miR159a were the best genes when the leaves were treated by methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA), respectively. Our results provide suitable reference genes for future investigations on miRNA functions in tea plants. PMID:27240406

  16. Selection and Verification of Candidate Reference Genes for Mature MicroRNA Expression by Quantitative RT-PCR in the Tea Plant (Camellia sinensis

    Directory of Open Access Journals (Sweden)

    Hui Song

    2016-05-01

    Full Text Available Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is a rapid and sensitive method for analyzing microRNA (miRNA expression. However, accurate qRT-PCR results depend on the selection of reliable reference genes as internal positive controls. To date, few studies have identified reliable reference genes for differential expression analysis of miRNAs among tissues, and among experimental conditions in plants. In this study, three miRNAs and four non-coding small RNAs (ncRNA were selected as reference candidates, and the stability of their expression was evaluated among different tissues and under different experimental conditions in the tea plant (Camellia sinensis using the geNorm and NormFinder programs. It was shown that miR159a was the best single reference gene in the bud to the fifth leaf, 5S rRNA was the most suitable gene in different organs, miR6149 was the most stable gene when the leaves were attacked by Ectropis oblique and U4, miR5368n and miR159a were the best genes when the leaves were treated by methyl jasmonate (MeJA, salicylic acid (SA and abscisic acid (ABA, respectively. Our results provide suitable reference genes for future investigations on miRNA functions in tea plants.

  17. Viral etiology of respiratory infections in children in southwestern Saudi Arabia using multiplex reverse-transcriptase polymerase chain reaction

    OpenAIRE

    Al-Ayed, Mohamed S.; Asaad, Ahmed M; Qureshi, Mohamed A.; Ameen, Mohammed S.

    2014-01-01

    Objectives: To investigate 15 respiratory viruses in children with acute respiratory tract infections (ARTIs) using multiplex reverse-transcriptase polymerase chain reaction (RT-PCR), and to analyze the clinical and epidemiological features of these viruses. Methods: In a cross-sectional study, 135 children, ≤5 years of age who presented with ARTIs in Najran Maternity and Children Hospital, Najran, Saudi Arabia between October 2012 and July 2013 were included. The clinical and sociodemographi...

  18. Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Abdul Wajid, Muhammad Wasim, Tahir Yaqub, Shafqat F Rehmani, Tasra Bibi, Nadia Mukhtar, Javed Muhammad, Umar Bacha, Suliman Qadir Afridi, Muhammad Nauman Zahid, Zia u ddin, Muhammad Zubair Shabbir, Kamran Abbas & Muneer Ahmad ### Abstract In the present protocol we describe the real time reverse transcription polymerase chain reaction (rRT-PCR) assay for the rapid detection/pathotyping of Newcastle disease virus (NDV) isoaltes in clinical samples. Fusion gene and matrix ...

  19. Detection of colonic cells in peripheral blood of colorectal cancer patients by means of reverse transcriptase and polymerase chain reaction.

    OpenAIRE

    Castells, A.; Boix, L.; Bessa, X; Gargallo, L.; Piqué, J M

    1998-01-01

    Circulating tumour cells play a central role in the metastatic process, but little is known about the relationship between this cellular subpopulation and the development of secondary disease. This study was aimed at assessing the presence of colonic cells in peripheral blood of patients with colorectal cancer in different evolutionary stages, by means of reverse transcriptase polymerase chain reaction (RT-PCR) targeted to carcinoembryonic antigen (CEA) mRNA. In vitro sensitivity was establis...

  20. 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

  1. Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

    Institute of Scientific and Technical Information of China (English)

    Zhu Yang; Guoliang Mao; Yujun Liu; Yuan-Chuan Chen; Chengjing Liu; Jun Luo; Xihan Li

    2013-01-01

    A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N 1/2009 influenza A virus.In this study,we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus.The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers.The qRT-PCR assays with the newly designed primers are highly specific,and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human,swine,and raccoon dog origin.Furthermore,the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction,respectively.When tested with 83 clinical samples,32 were detected to be positive using the qRT-PCR assays with our designed primers,while only 25 were positive by the assays with the WHO-recommended primers.These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

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

    OpenAIRE

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

    2004-01-01

    Abstract Background Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real ...

  3. Polymerase chain reaction

    OpenAIRE

    Gaurav Solanki

    2015-01-01

    The polymerase chain reaction (PCR) is a technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. There are three major steps involved in the PCR technique: denaturation, annealing and extension. PCR is useful in the investigation...

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

    Science.gov (United States)

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

    2016-05-01

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

  5. Real time polymerase chain reaction in diagnosis of chronic myeloid leukemia

    International Nuclear Information System (INIS)

    Objective: To compare the sensitivity and specificity of Real Time Polymerase Chain Reaction (RT-PCR) with conventional cytogenetics in diagnosis of chronic myeloid leukemia. Study Design: A cross-sectional, analytical study. Place and Duration of Study: The Armed Forces Institute of Pathology (AFIP), Rawalpindi, from December 2010 to January 2012. Methodology: A total number of 40 patients were studied, in which all were diagnosed as CML on peripheral blood and bone marrow aspiration. The subjects were tested for the presence of Philadelphia (Ph) chromosome by cytogenetics and BCR-ABL fusion gene by RT-PCR. 2-3 ml of venous blood was collected, half in sodium heparin (anti-coagulant) for cytogenetics and half in EDTA for PCR. For cytogenetics, cells were cultured for 72 hours in RPMI 1640 medium and examined by arresting in metaphase using Colchicine to identify Philadelphia chromosome. For PCR, RNA extraction was done by Tri Reagent LS (MRC, USA) and cDNA was synthesized using reverse transcriptase and gene specific primer. RT- PCR was done on ABI-7500. The positive samples were identified when fluorescence exceeded threshold limit. Results of cytogenetics and RT PCR were compared. Results: Out of the 40 patients, PCR showed 37 (92.5%) were positive and 3 (7.5%) were negative for BCR-ABL fusion gene, whereas in cytogenetics 28 (70%) were positive for Ph chromosome and 12 (30%) were negative for Ph chromosome. Sensitivity and specificity of cytogenetics was 75.6% and 100% respectively. Conclusion: Real time PCR as compared to cytogenetics is less tedious, gives quick results, does not require multiple sampling due to culture failure and can be done on peripheral blood. (author)

  6. DETECTION OF BREAST CANCER MICROMETASTASES IN BONE MARROW USING REVERSE-TRANSCRIPTASE CHAIN REACTION AND SOUTHERN HYBRIDIZATION

    Institute of Scientific and Technical Information of China (English)

    LI Jin-feng; ZHANG Lei; SUN Su-lian

    1999-01-01

    Objective: The aim of this study was to detect micrometastases in bone marrow of primary breast cancer patients, and compare with other clinical parameters.Methods: Cytokeratin 19 (CK-19) gene mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization.Human breast cancer cell line T47D was mixed with bone marrow cells in different proportions. The positive detection rate was compared among RT-PCR, Southern blotting and immunohistochemistry (IHC) methods. Results: Cytokeratin 19 gene was expressed in all 6 positive control samples while the expression was not seen in 8 negative control samples. In all 54 patients 14 cases were CK-19 positive (25.9%) by RT-PCR, another positive signal was obtained in 5/54 (9.3%) of bone marrow samples by Southern blotting. The total positive cases are 19/54 (35.2%).CK-19 IHC+ cells were detected at a dilution of one T47D cell in 5×104 bone marrow cells, while the sensitivity detected by PCR and Southern blot hybridization was at 1:5×105 and 1:1×106, respectively. This demonstrates that RT-PCR and Southern blotting was at least 20 times more sensitive than the IHC method. The micrometastases positive rate of the larger tumor size group (>5.0 cm) was significantly (P<0.05) greater than that of the smaller tumor size group (0-2.0 cm). Conclusion: detection of micrometastases in bone marrow by RT-PCR and Southern blotting, using CK-19 as a biological marker, is highly sensitive and it is a method to be used for anticipating the prognosis of breast cancer patients.

  7. DNA microarray-based solid-phase RT-PCR for rapid detection and identification of influenza virus type A and subtypes H5 and H7

    DEFF Research Database (Denmark)

    Yi, Sun; Dhumpa, Raghuram; Bang, Dang Duong;

    2011-01-01

    article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification of......Endemic of avian influenza virus (AIV) in Asia and epizootics in some European regions have caused considerable public concern on a possible pandemic of AIV. A rapid method for virus detection and effective surveillance in wild avian, poultry production as well as in humans is required. In this...... RNA extract in the liquid phase with sequence-specific nested PCR on the solid phase. A simple ultraviolet cross-linking method was used to immobilize the DNA probes over an unmodified glass surface, which makes solid-phase PCR a convenient possibility for AIV screening. The testing of 33 avian fecal...

  8. Evaluation of two real-time polymerase chain reaction assays for Porcine epidemic diarrhea virus (PEDV) to assess PEDV transmission in growing pigs.

    Science.gov (United States)

    Miller, Laura C; Crawford, Kimberly K; Lager, Kelly M; Kellner, Steven G; Brockmeier, Susan L

    2016-01-01

    In April 2013, a Porcine epidemic diarrhea virus (PEDV) epidemic began in the United States. As part of the response, real-time reverse transcription polymerase chain reaction (RT-PCR) assays to detect PEDV were developed by several veterinary diagnostic laboratories. Our study evaluated RT-PCR PEDV assays that detect the N gene (gN) and S gene (gS) for their ability to detect PEDV infection and the transmission potential of pigs experimentally exposed to PEDV. Detection limits and quantification cycle (Cq) values of real-time RT-PCR were assayed for PEDV samples and positive controls for both gN and gS. The limit of detection for the gN assay was 10(-6) (mean Cq: 39.82 ± 0.30) and 10(-5) (mean Cq: 39.39 ± 0.72) for the gS assay with PEDV strain USA/Colorado/2013. Following recommended guidelines, rectal swabs (n = 1,064) were tested; 354 samples were positive by gN assay and 349 samples were positive by gS assay (Cq ≤ 34.99), 710 samples were negative by gN assay and 715 were negative by gS assay (Cq > 34.99) of which 355 and 344 were "undetermined" (i.e., undetected within a threshold of 40 RT-PCR cycles, by gN and gS assays, respectively). The coefficient of variation (intra-assay variation) ranged from 0.00% to 2.65% and interassay variation had an average of 2.75%. PEDV could be detected in rectal swabs from all pigs for ~2 weeks postinfection at which time the prevalence began to decrease until all pigs were RT-PCR negative by 5 weeks postinfection. Our study demonstrated that RT-PCR assays functioned well to detect PEDV and that the gN assay was slightly better. PMID:26699519

  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. RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza

    DEFF Research Database (Denmark)

    Dybkær, Karen; Munch, Mette; Handberg, Kurt;

    2003-01-01

    low-pathogenicity AIV, from wild aquatic birds, and from domestic ducks. The AIV was detected in 32 swab pools by RT-PCR-ELISA compared to 23 by virus isolation (VI) in embryonated specific pathogen free (SPF) chicken eggs. Thus, 39% more specimens were positive by RT-PCR-ELISA than by VI. Two of the...

  11. 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

  12. Rapid diagnosis of avian influenza virus in wild birds: Use of a portable rRT-PCR and freeze-dried reagents in the field

    Science.gov (United States)

    Takekawa, J.Y.; Hill, N.J.; Schultz, A.K.; Iverson, S.A.; Cardona, C.J.; Boyce, W.M.; Dudley, J.P.

    2011-01-01

    Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal. Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene. The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey

  13. Detection of classical swine fever virus (CSFV) in clinical samples by RT-PCR assay in clinical samples by RT-PCR assay using different pairs of primers

    International Nuclear Information System (INIS)

    The aim was to compare the efficiency of RT-PCT assays using four pairs of primers selected from different regions of the CSFV genome for the detection of CSFV in clinical samples of swine and wild boars. The four RT-PCR assays were able to detect CSFV in all 20 clinical samples which had been collected from dead swine and wild boars during the outbreaks of CSF in Slovakia in 1993 and 1994. The quality of the selected RT-PCR primers was determined as follows: gp55L/gp55U (E2), 324/326 (5'-NC), S1/S2 (NS5B) and gp54L/gp54U (NS2 genomic region). We conclude that gp55L/gp55U primers are the most suitable for direct detection of CSFV by RT-PCR in tissue homogenates of diseased animals

  14. Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Gaurav Solanki

    2012-10-01

    Full Text Available The polymerase chain reaction (PCR is a technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. There are three major steps involved in the PCR technique: denaturation, annealing and extension. PCR is useful in the investigation and diagnosis of a growing number of diseases. PCR is also used in forensics laboratories. PCR can identify genes that have been implicated in the development of cancer. The present paper is an attempt to review basics of PCR in relation to its methods, application and use.

  15. Optimizing polymerase chain reaction testing for the diagnosis of pertussis: current perspectives

    Directory of Open Access Journals (Sweden)

    Arbefeville S

    2015-09-01

    Full Text Available Sophie Arbefeville, Patricia Ferrieri Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, MN, USA Abstract: Nucleic acid testing has revolutionized the diagnosis of pertussis in the clinical microbiology laboratory and has become the main avenue of testing for pertussis infection. Real-time polymerase chain reaction (RT-PCR is an important tool for timely diagnosis of pertussis and is more sensitive than culture. The most commonly amplified targets are the insertion-sequence (IS genes, which are found in multiple copies in the genome of Bordetella species. Some strains of Bordetella pertussis have more than 200 copies of IS481 in their genome. This high number of repeats allows RT-PCR assays to be very sensitive and makes nucleic acid testing two to three times more sensitive than culture. Despite these advantages, RT-PCR can give inaccurate results due to contamination or lack of specificity. Contamination can easily happen during specimen collection, DNA extraction, or nucleic acid amplification steps. To avoid contamination, laboratories need to have quality controls and good workflows in place. The poor specificity of the nucleic acid assays amplifying the IS genes is because they are found in various Bordetella species and, thus, not unique to a specific species. Bordetella holmesii, a more recently described Bordetella species found to be responsible for respiratory symptoms similar to pertussis in adolescents and adults, can be misidentified as B. pertussis in RT-PCR assays that amplify only the IS481 target. Use of multiple targets may improve specificity of RT-PCR assays for pertussis. In the past few years, the US Food and Drug Administration has cleared three commercial assays for the detection of B. pertussis in respiratory specimens. Several commercial assays and analyte-specific reagents, which are not US Food and Drug Administration cleared, are available for the detection of one

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

    Science.gov (United States)

    Tang, Yi; Lu, Huaguang

    2016-04-01

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

  17. Detection of PERV by polymerase chain reaction and its safety in bioartificial liver support system

    Institute of Scientific and Technical Information of China (English)

    Hai-Hui Wang; Ying-Jie Wang; Hong-Ling Liu; Jun Liu; Yan-Ping Huang; Hai-Tao Guo; Yu-Ming Wang

    2006-01-01

    AIM: To establish a method detecting porcine endogenous retrovirus (PERV) in China experimental minipigs and to evaluate the safety of PERV in three individuals treated with bioartificial liver support systems based on porcine hepatocytes.METHODS: Porcine hepatocytes were isolated with two-stage perfusion method, then cultured in the bioreactor, which is separated by a semipermeable membrane (0.2μm) from the lumen through which the patients' blood plasma was circulated. After post-hemoperfusion, patients' blood was obtained for screening. Additionally, samples of medium collected from both intraluminal and extraluminal compartments of the laboratory bioreactor and culture supernate in vitro was analyzed. The presence of viral sequences was estimated by polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, the infection of virus in the supernate of common culture was ascertained by exposure to the fetal liver cells.RESULTS: PERV-specific gag sequences were found in the porcine hepatocytes using RT-PCR. and were detected in all samples from the intraluminal,extraluminal samples and culture supernate. However,culture supernatant from primary porcine hepatocytes (cleared of cellular debris) failed to infect human fetal liver cells. Finally, RT-PCR detected no PERV infection was found in the blood samples obtained from three patients at various times post-hemoperfusion.CONCLUSION: The assays used are specific and sensitive, identified by second PCR. PERVs could be released from hepatocytes cultured in bioreactor without the stimulation of mitogen and could not be prevented by the hollow fiber semipermeable membrane, indicating the existence of PERV safety in extracorporeal bioartificial liver support system (EBLSS).

  18. Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

    Directory of Open Access Journals (Sweden)

    Alyne Moraes Costa

    2013-02-01

    Full Text Available ELISA in situ can be used to titrate hepatitis A virus (HAV particles and real-time polymerase chain reaction (RT-PCR has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5 copies/mL (p = 0.0002, while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001. At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

  19. Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens

    OpenAIRE

    Mani Revathy; Kulandai Lily Therese; Radhakishnan Bagyalakshmi; Chokaliingam Chandrasekar; Suria Kumar; Madhavan, Hajib N.

    2014-01-01

    Background & objectives: As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - staining technique, application of a real time polymerase chain reaction (RT-PCR) targeting kex 1 gene and application of nested PCR targeting mitochondrial large subunit (mtLSU) gene for rapid detection of...

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

  2. Análise de citocinas pela RT-PCR em pacientes com rinite alérgica RT-PCR cytokine study in patients with allergic rhinitis

    Directory of Open Access Journals (Sweden)

    Tarcimara Moreira da Silva

    2009-02-01

    Full Text Available Rinite alérgica é uma doença que decorre de um processo inflamatório da mucosa nasal conseqüente à reação de hipersensibilidade a alérgenos inalatórios e, eventualmente, alimentares. É mediada por IgE, envolvendo diferentes células, mediadores e citocinas. OBJETIVO: Avaliar as transcrições para as seguintes citocinas: IL-4, IL-5, IL-8 e IFN-gama, particularmente importantes no processo alérgico nasal, principalmente IL-4 e IL-5. Neste estudo, optou-se por avaliar os pacientes atópicos fora das crises alérgicas, com a finalidade de se conhecer as expressões das citocinas neste período. MATERIAL E MÉTODO: Realizou-se um estudo transversal e prospectivo, selecionando-se 30 pacientes, sendo 13 pacientes portadores de rinite alérgica paucissintomáticos e 17 pacientes não-atópicos. Os grupos foram selecionados através da história, do exame clínico otorrinolaringológico e do teste alérgico cutâneo - Prick Test. O perfil das citocinas foi pesquisado nos fragmentos de mucosa nasal, através da RT-PCR semiquantitativa, escolhida por apresentar boa reprodutibilidade e especificidade, utilizando-se como referência o gene da Beta-actina. RESULTADOS: Os valores de IL-5, IL-8, IFN-gama mantiveram-se homogêneos em relação ao grupo controle. A IL-4 apresentou diferença com significância estatística. CONCLUSÃO: Os pacientes alérgicos paucissintomáticos apresentaram normalização da expressão das citocinas na mucosa nasal à exceção de IL-4.Allergic rhinitis is an inflammatory reaction of the nasal mucosa, in consequence of an IgE mediated hypersensitive reaction to inhaling allergens, involving different mediators and cytokine cells. AIM: The purpose of this study was to evaluate the transcriptions for IL-4, IL-5, IL-8 and IFN-gama, particularly important in the nasal allergy process, especially IL-4 and IL-5. For this study we decided to evaluate atopic patients who were free from allergic crises, with the purpose of

  3. Improvement of a real-time RT-PCR assay for the detection of enterovirus RNA

    OpenAIRE

    Bruynseels Peggy; Verstrepen Walter A; Piqueur Marian AC; Mertens An H

    2009-01-01

    Abstract We describe an improvement of an earlier reported real-time RT-PCR assay for the detection of enterovirus RNA, based on the 5' exonuclease digestion of a dual-labeled fluorogenic probe by Taq DNA polymerase. A different extraction method, real-time RT-PCR instrument and primer set were evaluated. Our data show that the optimized assay yields a higher sensitivity and reproducibility and resulted in a significant reduced hands-on time per sample.

  4. Improvement of a real-time RT-PCR assay for the detection of enterovirus RNA

    Directory of Open Access Journals (Sweden)

    Bruynseels Peggy

    2009-07-01

    Full Text Available Abstract We describe an improvement of an earlier reported real-time RT-PCR assay for the detection of enterovirus RNA, based on the 5' exonuclease digestion of a dual-labeled fluorogenic probe by Taq DNA polymerase. A different extraction method, real-time RT-PCR instrument and primer set were evaluated. Our data show that the optimized assay yields a higher sensitivity and reproducibility and resulted in a significant reduced hands-on time per sample.

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

    Science.gov (United States)

    Papayiannis, Lambros C

    2014-02-01

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

  6. Validation of artificial microRNA expression by poly(A) tailing-based RT-PCR

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Rui Shi, Chenmin Yang, Ronald Sederoff & Vincent Chiang ### Abstract Here we describe a protocol for validating expression of artificial microRNAs (amiRNAs) by poly(A) tailing-based RT-PCR. Total RNAs, including amiRNA, are poly(A) tailed using E.coli. poly(A) polymerase. Poly(A) tailed amiRNA can be converted into cDNA along with mRNAs in a reverse transcription reaction primed by a standard poly(T) anchor adaptor. AmiRNA can then be amplified and quantitated by real-tim...

  7. COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

    Directory of Open Access Journals (Sweden)

    Vasila Packeer Mohamed

    2014-05-01

    Full Text Available ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively large amount of samples was involved. Total RNA was extracted using Total RNA purification kit (without DNase treatment; Norgen, Canada and RNeasy mini kit (with DNase treatment; Qiagen, USA respectively. The extracted RNA was then reverse transcribed, and the cDNA was subjected to PCR-amplifying 18S. Yield from RNeasy kit was significantly higher (0.316 ± 0.033 µg/µl; p=0.004 than Total RNA purification kit (0.177 ± 0.0243 µg/µl. However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. Total RNA purification kit is less expensive than RNeasy kit. Since there is no DNase treatment step in the former, extraction time for RNA is shorter. When the extracted RNA was subjected to RT-PCR, both methods were able to show detection of 18S at 219 bp.   Therefore, this study demonstrates that both protocols are suitable for RNA extraction for CHO-K1 cells. RNeasy mini kit (Qiagen is recommended if higher yields is the primary concern and Total RNA Purification kit (Norgen is recommended if time and cost are concerned. ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat.  Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil.  Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem.  Tujuan kajian dijalankan

  8. Quantitative Real-time Polymerase Chain Reaction for Enteropathogenic Escherichia coli: A Tool for Investigation of Asymptomatic Versus Symptomatic Infections

    Science.gov (United States)

    Barletta, Francesca; Mercado, Erik; Ruiz, Joaquim; Ecker, Lucie; Lopez, Giovanni; Mispireta, Monica; Gil, Ana I.; Lanata, Claudio F.; Cleary, Thomas G.

    2011-01-01

    Background. Enteropathogenic Escherichia coli (EPEC) strains are pediatric pathogens commonly isolated from both healthy and sick children with diarrhea in areas of endemicity. The aim of this study was to compare the bacterial load of EPEC isolated from stool samples from children with and without diarrhea to determine whether bacterial load might be a useful tool for further study of this phenomenon. Methods. EPEC was detected by polymerase chain reaction (PCR) of colonies isolated on MacConkey plates from 53 diarrheal and 90 healthy children aged <2 years. DNA was isolated from stool samples by cetyltrimethylammonium bromide extraction. To standardize quantification by quantitative real-time PCR (qRT-PCR), the correlation between fluorescence threshold cycle and copy number of the intimin gene of EPEC E2348/69 was determined. Results. The detection limit of qRT-PCR was 5 bacteria/mg stool. The geometric mean load in diarrhea was 299 bacteria/mg (95% confidence interval [CI], 77–1164 bacteria/mg), compared with 29 bacteria/mg (95% CI, 10–87 bacteria/mg) in control subjects (P = .016). Bacterial load was significantly higher in children with diarrhea than in control subjects among children <12 months of age (178 vs 5 bacteria/mg; P = .006) and among children with EPEC as the sole pathogen (463 vs 24 bacteria/mg; P = .006). Conclusions. EPEC load measured by qRT-PCR is higher in diarrheal than in healthy children. qRT-PCR may be useful to study the relationship between disease and colonization in settings of endemicity. PMID:22028433

  9. Longitudinal study of the detection of Bluetongue virus in bull semen and comparison of real-time polymerase chain reaction assays.

    Science.gov (United States)

    Gu, Xingnian; Davis, Rodney J; Walsh, Susan J; Melville, Lorna F; Kirkland, Peter D

    2014-01-01

    Infection with Bluetongue virus (BTV) is a significant impediment to the global movement of bovine semen. Repeat testing of blood from donor animals is specified in the World Organization for Animal Health (OIE) Manual for the export of semen from regions where BTV may be present. Screening of blood or semen samples has usually been carried out by virus isolation (VI) either by inoculation of chicken embryos followed by passage onto insect and mammalian cell cultures or in vivo inoculation of sheep followed by serology to detect seroconversion. Direct testing of semen for BTV would enable earlier release of semen samples and avoid repeat testing of the donor, as well as provide an option for releasing batches of semen that were collected without certification of the donor. Quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) assays overcome most of the limitations of other methods and have the potential to provide higher sensitivity. The present study compared 5 qRT-PCR assays, including 2 commercially available kits, for the detection of BTV in semen serially collected from 8 bulls over a period of 90 days after experimental infection. The results of the study show that at least one of the qRT-PCR assays is extremely reproducible and has both very high sensitivity and specificity to reliably detect all available serotypes. The preferred qRT-PCR gave consistently superior results to VI, sheep inoculation, and conventional RT-PCR. Therefore, the assay can be recommended for the screening of bovine semen for freedom from BTV. PMID:24532692

  10. Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens

    Directory of Open Access Journals (Sweden)

    Mani Revathy

    2014-01-01

    Full Text Available Background & objectives: As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - staining technique, application of a real time polymerase chain reaction (RT-PCR targeting kex 1 gene and application of nested PCR targeting mitochondrial large subunit (mtLSU gene for rapid detection of P. jirovecii in HIV positive patients. Methods: One hundred and fifty sputum specimens from HIV positive (n = 75 and HIV negative (n = 75 patients were subjected to three different techniques -KOH/Calcoflour and Grocott methanamine silver staining (GMS, RT-PCR targeting kex1 gene, PCR targeting mtLSU region followed by DNA sequencing and BLAST analysis. Results: Among the 75 HIV positive patients, P. jirovecii was detected in 19 (25.33% patients by the staining techniques, and in 23 (30.65% patients each by PCR targeting mtLSU region and by RT- PCR targeting kex1 gene of P. jirovecii. PCR based DNA sequencing targeting mtLSU region revealed 97-100 per cent sequence homology with P. jirovecii sequences in GenBank. Interpretation & conclusions: Of the three techniques for detection of P. jirovecii evaluated in this study, false negativity was found to be more in staining technique and it also required high technical expertise to interpret the result. Both nested PCR and RT-PCR were reliable and equally sensitive, in rapid detection of P. jirovecii, but RT-PCR technique also generated the copy numbers for knowing the severity of infection.

  11. Real-time ed end-point Polymerase Chain Reaction per la quantizzazione del DNA di Citomegalovirus: confronto tra metodi e con il test per l’antigene pp65

    Directory of Open Access Journals (Sweden)

    Tiziano Allice

    2006-03-01

    Full Text Available Quantitave Polymerase Chain Reaction (PCR for Cytomegalovirus (CMV DNA provides highly sensitive and specific data for detecting CMV as well as monitoring the infection and determining the appropriate antiviral strategy.To determine the clinical application of a recently introduced real-time (RT PCR assay for CMV DNA quantitation in peripheral blood leukocytes (PBLs and defining its correlation with the commercial quantitative end-point (EP PCR method COBAS AMPLICOR CMV Monitor and pp65 antigen test. Sequential PBL samples (n=158 from 32 liver transplanted patients with CMV asymptomatic infection and positive for CMV DNA by EP-PCR were retrospectively analysed with RT-PCR and studied according to pp65 antigen levels. A good correlation was found between RT-PCR and pp65 antigen test (r=0.691 and between the two PCR assays (r=0.761. RT-PCR data were significantly higher in pre-emptive treated patients (those with >20 pp65+positive cells, median value: 3.8 log10 copies/500,000 PBLs than in not-treated ones (2.9 logs.According to pp65 levels of 0, 1-10, 11-20, 21-50, 51-100 and >100 positive cells/200,000 PBLs, median CMV DNA load by RT-PCR was 2.6, 3.0, 3.6, 4.0. 4.2 and 4.8, log10 copies/ 500,000 PBLs, respectively (EP-PCR CMV DNA levels: 2. 8, 2.9, 3.8, 3.7, 3.9 and 4.0 logs. For samples with >20 pp65+cells, that is above the level at which pre-emptive therapy was started, RT-PCR values were significantly higher than in groups with less than 20 pp65+cells, whereas EP-PCR values did not significantly differ and showed a slower progression rate. Dilutions of DNA from CMV AD169 strain were used to probe RT-PCR reproducibility (between and intra-assay variability < 2% and sensitivity (100% detection rate at 10 copies/reaction, 28.5% with EP-PCR. A significant improvement is coming from the introduction of RT-PCR to the study of CMV DNA dynamics in differently CMV infected patients due to a more reliable quantitation of CMV DNA for moderate and high

  12. Outcome on EPIZONE Extension on VER/ VNN: Diagnostics, proficiency test and qRT-PCR validation

    DEFF Research Database (Denmark)

    Bigarré, Laurent; Panzarin, Valentina; Baud, Marine;

    2012-01-01

    : RNA1 and RNA2. Till now, only one universal real-time RT-PCR method has been published which detects all variants of RNA2 (Panzarin et al., 2010) in clinical cases. An additional method based on detection of RNA1 would be useful to confirm results obtained from RNA2. Another advantage of detecting RNA...... test for detection of aquatic nodaviruses by real time RT-PCR targeting RNA1 and RNA2, respectively. - To test a newly developed real-time RT-PCR targeting RNA1: sensitivity, specificity, range of detection and genetic information provided by sequencing the PCR product. Materials & methods Primers and...... extracted by each partner. In the meantime, ANSES produced and distributed RNA extracted from healthy or infected fish (2 genogroups), or cell culture; one sample from cell culture had to be serially diluted to test the sensitivity of each method in partners’ hands. Samples were tested in duplicates and the...

  13. Detection and identification of infectious bronchitis virus by RT-PCR in Iran.

    Science.gov (United States)

    Homayounimehr, Alireza; Pakbin, Ahmad; Momayyez, Reza; Fatemi, Seyyedeh Mahsa Rastegar

    2016-06-01

    Infectious bronchitis virus (IBV) causes severe diseases in poultry with significant economic consequences to the poultry industry in Iran. The aim of this study was the detection and identification of IBV by reverse transcription(RT)-PCR in Iran. Ten IB virus strains were detected by testing trachea, cecal tonsil, and kidney tissues collected from broiler and layer farms in Iran. In order to detect infectious bronchitis virus, an optimized RT-PCR was used. Primers targeting the conserved region of known IBV serotypes were used in the RT-PCR assay. Primers selectively detecting Massachusetts and 793/B type IB viruses were designed to amplify the S1 gene of the virus and used in the nested PCR test. Our findings indicate the circulation of at least three genotypes of IB viruses (Massachusetts, 793/B, and variant 2) among poultry flocks. PMID:27010714

  14. Real-Time Polymerase Chain Reaction as a Tool for Evaluation of Magnetic Poly(Glycidyl methacrylate)-Based Microspheres in Molecular Diagnostics.

    Science.gov (United States)

    Trachtová, Stepánka; Spanová, Alena; Horák, Daniel; Kozáková, Hana; Rittich, Bohuslav

    2016-01-01

    DNA amplification by real-time polymerase chain reaction (RT-PCR) was used for the evaluation of efficiency of polymer coating of magnetic hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) and poly(glycidyl methacrylate) (PGMA) microspheres with/without carboxyl groups. The inhibition effect of magnetic microspheres on real-time polymerase chain reaction (RT-PCR) course was evaluated by regression analysis after the addition of different concentrations of tested microspheres to PCR mixtures. Microspheres mostly did not interfere in RT-PCR till the concentration 50 µg/25 µl PCR mixture. No relationship between Fe content (and microsphere diameter) and inhibition effect was found. Microspheres containing carboxyl groups extinguished the fluorescence at lower concentrations (10-20 µg/25 µl PCR mixture) without inhibition of DNA amplification as PCR products were detected using agarose gel electrophoresis. Negative effect of maghemite on PCR course was partially reduced by coating of magnetic core by silica or polymers. Two inhibition mechanisms of DNA amplification were discussed in this work. PMID:26708828

  15. Sensitive detection of Renibacterium salmoninarum in whole fry, blood, and other tissues of pacific salmon by reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Rhodes, L D; Nilsson, W B; Strom, M S

    1998-12-01

    A sensitive, reproducible assay for detecting Renibacterium salmoninarum in a variety of tissues, including blood, has been developed. This assay, based on reverse transcription-polymerase chain reaction (RT-PCR) of 16S ribosomal RNA, exhibited sensitivity to RT-PCR make it a useful tool for both research and husbandry applications. PMID:9892717

  16. 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

    OpenAIRE

    Wang Wei; Li Xiyan; Yang Lei; Li Changgui; Zou Shumei; Shao Ming; Wen Leying; Gao Yan; Gao Rongbao; Shu Yuelong

    2011-01-01

    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 o...

  17. Supply chain reaction

    International Nuclear Information System (INIS)

    'Logic' (Leading Oil and Gas Industry Competitiveness) is a government-industry supply chain management initiative which aims to improve the competitiveness of the UK's North Sea business by 1 billion UK pounds by 2002 and its export performance by 50% inside 5 years. Much of the article is devoted to the background and views of Logic's chief executive Chris. Freeman. Freeman makes clear that 'unlike Crine, we are not a cost-reduction initiative: that may be one of the outcomes, but we are really focusing on the co-operation side of things'. Logic aims to change the culture of the UK offshore industry through example. Freeman believes that the creation of collaborative success will flag up industry and give credence to Logics objectives

  18. A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

    Science.gov (United States)

    Tolardo, Aline Lavado; de Souza, William Marciel; Romeiro, Marilia Farignoli; Vieira, Luiz Carlos; Luna, Luciano Kleber de Souza; Henriques, Dyana Alves; de Araujo, Jansen; Siqueira, Carlos Eduardo Hassegawa; Colombo, Tatiana Elias; Aquino, Victor Hugo; da Fonseca, Benedito Antonio Lopes; Bronzoni, Roberta Vieira de Morais; Nogueira, Maurício Lacerda; Durigon, Edison Luiz; Figueiredo, Luiz Tadeu Moraes

    2016-01-01

    Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections. PMID:27276185

  19. Molecular detection of Papaya meleira virus in the latex of Carica papaya by RT-PCR

    NARCIS (Netherlands)

    Araujo, de M.M.M.; Tavares, E.T.; Silva, da F.R.; Marinho, V.L.D.; Souza, M.T.

    2007-01-01

    A RT-PCR assay was developed for early and accurate detection of Papaya meleira virus (PMeV) in the latex from infected papayas. The meleira disease is characterized by an excessive exudation of more fluidic latex from fruits, leaves and stems. This latex oxidises and gives the fruit a ¿sticky¿ text

  20. Different expression of house-keeping genes by quantitative real time RT-PCR

    Czech Academy of Sciences Publication Activity Database

    Ullmannová, V.; Truksa, Jaroslav; Haškovec, C.; Kovář, Jan

    CR: XXX , 2003, s. 1. [Functional genomics and diseases. Praha (CZ), 05.05.2003-07.05.2003] Institutional research plan: CEZ:AV0Z5052915 Keywords : quantitative real time RT-PCR * house-keeping genes expression Subject RIV: EB - Genetics ; Molecular Biology

  1. 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. PMID:27036502

  2. Detection panel for identification of twelve hemorrhagic viruses using real-time RT-PCR

    Czech Academy of Sciences Publication Activity Database

    Fajfr, M.; Neubauerová, V.; Pajer, P.; Kubíčková, P.; Růžek, Daniel

    2014-01-01

    Roč. 63, č. 3 (2014), s. 232-238. ISSN 1210-7913 Institutional support: RVO:60077344 Keywords : hemorrhagic fever * filovirus * arenavirus * real-time * RT-PCR – detection Subject RIV: EE - Microbiology, Virology Impact factor: 0.353, year: 2014

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

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

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

    Institute of Scientific and Technical Information of China (English)

    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.

  6. Detecting the Presence of Nora Virus in "Drosophila" Utilizing Single Fly RT-PCR

    Science.gov (United States)

    Munn, Bethany; Ericson, Brad; Carlson, Darby J.; Carlson, Kimberly A.

    2015-01-01

    A single fly RT-PCR protocol has recently been developed to detect the presence of the persistent, horizontally transmitted Nora virus in "Drosophila." Wild-caught flies from Ohio were tested for the presence of the virus, with nearly one-fifth testing positive. The investigation presented can serve as an ideal project for biology…

  7. Rapid and sensitive electrochemiluminescence detection of rotavirus by magnetic primer based reverse transcription-polymerase chain reaction

    Energy Technology Data Exchange (ETDEWEB)

    Zhan Fangfang; Zhou Xiaoming [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China); Xing Da, E-mail: xingda@scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China)

    2013-01-25

    Graphical abstract: In this work, we have developed and demonstrated a magnetic primer based RT-PCR assay for ECL detection of rotavirus. In the presence of two functional primers (magnetic primer and TBR-primer) and PCR reagents, cDNA from RT was amplified directly onto MPs during PCR cycles of denaturation, annealing and extension. The resulting MPs-TBR complexes were easily loaded on the electrode surface and produced a concentrated ECL signal. The figure shows the schematic illustration of magnetic primer RT-PCR based ECL assay for rotavirus detection. Highlights: Black-Right-Pointing-Pointer A novel method for detection of rotavirus has been developed. Black-Right-Pointing-Pointer In the presence of magnetic primer, TBR-primer and PCR reagents, cDNA form RT was amplified directly onto MPs. Black-Right-Pointing-Pointer To obtain the best sensing and efficient performance, important parameters associated with the efficiency were investigated carefully. Black-Right-Pointing-Pointer The proposed method will find numerous applications in food safety field and clinical diagnosis. - Abstract: A novel method for detection of rotavirus has been developed by integrating magnetic primer based reverse transcription-polymerase chain reaction (RT-PCR) with electrochemiluminescence (ECL) detection. This is realized by accomplishing RT of rotavirus RNA in traditional way and performing PCR of the resulting cDNA fragment on the surface of magnetic particles (MPs). In order to implement PCR on MPs and achieve rapid ECL detection, forward and reverse primers are bounded to MPs and tris-(2,2 Prime -bipyridyl) ruthenium (TBR), respectively. After RT-PCR amplification, the TBR labels are directly enriched onto the surface of MPs. Then the MPs-TBR complexes can be loaded on the electrode surface and analyzed by magnetic ECL platform without any post-modification or post-incubation process. So some laborious manual operations can be avoided to achieve rapid yet sensitive detection

  8. Expression Profile of IL-35 mRNA in Gingiva of Chronic Periodontitis and Aggressive Periodontitis Patients: A Semiquantitative RT-PCR Study

    Directory of Open Access Journals (Sweden)

    Nagaraj B. Kalburgi

    2013-01-01

    Full Text Available Background. Proinflammatory and anti-inflammatory cytokines play a key role in the pathogenesis of periodontal diseases. Secretion of bioactive IL-35 has been described by T regulatory cells ( and is required for their maximal suppressive activity. are involved in the modulation of local immune response in chronic periodontitis patients. Objective. Hence, the present study was aimed to investigate the expression of IL-35 mRNA in chronic periodontitis and aggressive periodontitis patients. Materials and Methods. The present study was carried out in 60 subjects, which included 20 chronic periodontitis patients, 20 aggressive periodontitis patients, and 20 periodontally healthy controls. IL-35 mRNA expression in gingival tissue samples of all subjects was semiquantitatively analyzed using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR. Results. The present study demonstrated the expression of IL-35 mRNA in gingival tissues of all the three groups. IL-35 mRNA expression was highest in chronic periodontitis subjects ( as compared to the aggressive periodontitis group ( and least seen in healthy patients (. Conclusion. The increased expression of IL-35 in chronic and aggressive periodontitis suggests its possible role in pathogenesis of periodontitis. Future studies done on large samples with intervention will strengthen our result.

  9. Expression of Somatostatin Receptor (SSTR) Subtypes (SSTR-1, 2A, 3, 4 and 5) in Neuroendocrine Tumors Using Real-time RT-PCR Method and Immunohistochemistry

    International Nuclear Information System (INIS)

    Molecule targeting therapy using somatostatin (SS) analogues has become a widely accepted modality to treat neuroendocrine tumors (NETs), particularly gastrointestinal (GI) and pancreatic endocrine tumors. On the other hand, little is known about the expression of somatostatin receptor (SSTR) subtypes in neuroendocrine carcinomas (NECs). We investigated the expression of SSTR subtypes (SSTR-1, 2A, 3, 4 and 5) using real-time reverse transcription polymerase chain reaction (RT-PCR) method and immunohistochemistry in 32 neuroendocrine neoplasms (9 NET G1, 2 NET G2, 18 NECs G3 and 3 mixed NEC G3) of various primary sites. Expression of more than two SSTR subtypes was detected in all neuroendocrine neoplasms examined. Expression of SSTR-2A mRNA was significantly higher than other subtypes. In addition, mRNA expression of SSTR-3 and SSTR-5 was significantly low or below the detection level except for gastroduodenal NET G1. No significant difference of the expression of SSTR subtypes was observed between the NET and NEC groups. The expression of protein and mRNA was generally well correlated. In conclusion, NECs would be a good candidate for molecule targeting therapy using SS analogues, and the expression of SSTR-2A can be useful as a biomarker of neuroendocrine differentiation. We have demonstrated that NEC G3 small cell type shows a different expression profile of SSTR subtypes compared with NET and NEC non-small cell type

  10. Establishment and Application of a TaqMan Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Rubella Virus RNA

    Institute of Scientific and Technical Information of China (English)

    Li-Hong ZHAO; Yu-Yan MA; Hong WANG; Shu-Ping ZHAO; Wei-Ming ZHAO; Hua LI; Lei-Yi WANG

    2006-01-01

    The aim of this study was to establish and apply a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for rubella virus (RV) RNA. First, the primer and TaqMan probe concentrations, as well as reaction temperatures were optimized to establish an efficient real-time quantitative RT-PCR assay for RV RNA. Next, an RV-specific PCR amplicon was made as an external standard to estimate the linearity, amplification efficiency, analytical sensitivity and reproducibility of the real time quantitative assay. Finally, the assay was applied to quantify RVRNA in clinical samples for rubella diagnosis.The RV-specific PCR amplicon was prepared for evaluation of the assay at 503 bp, and its original concentration was 2.75×109 copies/μl. The real time quantitative assay was shown to have good linearity (R2=0.9920), high amplification efficiency (E=1.91), high sensitivity (275 copies/ml), and high reproducibility (variation coefficient range, from 1.25% to 3.58%). Compared with the gold standard, the specificity and sensitivity of the assay in clinical samples was 96.4% and 86.4%, respectively. Therefore, the established quantitative RT-PCR method is a simple, rapid, less-labored, quantitative, highly specific and sensitive assay for RV RNA.

  11. Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process

    Directory of Open Access Journals (Sweden)

    Kohn Michael

    2007-10-01

    Full Text Available Abstract Background Since real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants. Results We directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/ Conclusion We present proof of the principle that a mechanistically based model can be fit to observations

  12. Application and evaluation of RT-PCR-ELISA for the nucleoprotein and RT-PCR for detection of low-pathogenic H5 and H7 subtypes of avian influenza virus

    DEFF Research Database (Denmark)

    Dybkær, Karen; Munch, Mette; Handberg, Kurt J.;

    2004-01-01

    -PCR-ELISA) and 2) the subtype specific RT-PCR for H5 and H7 were compared to the results obtained by inoculation of the same specimens into the allantoic cavity of embryonated specific pathogen free (SPF) hen's eggs. Using inoculation in SPF fowl eggs as standard the sensitivity of the NP RT-PCR-ELISA and the RT...

  13. In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

    International Nuclear Information System (INIS)

    Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in

  14. Rapid detection of sacbrood virus in honeybee using ultra-rapid real-time polymerase chain reaction.

    Science.gov (United States)

    Yoo, Mi-Sun; Thi, Kim Cuc Nguyen; Van Nguyen, Phu; Han, Sang-Hoon; Kwon, Soon-Hwan; Yoon, Byoung-Su

    2012-01-01

    A real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for the fast and highly sensitive detection of the sacbrood virus (SBV) genome and applied to honeybee samples. Using plasmid DNA containing a partial SBV genome and diluted serially, as few as 1×10(2)copies/μl (correlation co-efficiency >0.99) were detected by the qRT-PCR assay, whereas 1×10(3)copies/μl were detected by the conventional RT-PCR assay. As a rapid detection method, ultra-rapid real-time PCR (URRT-PCR) was carried out with a GenSpector TMC-1000 silicon-glass chip-based thermal cycler, which has a 6μl micro-chamber volume and a fast outstandingly heating/cooling rate. Using this method, 10(3)copies of pBX-SBV3.8 clone were detected within 17 min after 40 PCR cycles, including melting point analysis. To reduce the detection time for SBV, synthesis of the cDNA of the SBV genome from a honeybee sample was attempted for different reaction times and the cDNA was used as the template for URRT-PCR assays. The results indicated that a 5 min reaction time was sufficient to synthesize cDNA as the template for the SBV URRT-PCR assay. This study described a novel PCR-based method that is able to detect an RNA virus in environmental samples within 22 min, including reverse transcription, PCR detection and melting point analysis in real-time. PMID:22079620

  15. 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

    BACKGROUND: Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various...... 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...

  16. Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

    OpenAIRE

    Eduardo M. Del Aguila; Dutra, Marcio B; Silva, Joab T; Paschoalin, Vânia MF

    2005-01-01

    Background Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target. Results We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from ye...

  17. Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

    OpenAIRE

    Paschoalin Vânia MF; Silva Joab T; Dutra Marcio B; Del Aguila Eduardo M

    2005-01-01

    Abstract Background Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target. Results We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtaine...

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

    OpenAIRE

    HURLEY, JENNIFER M.; Dasgupta, Arko; Andrews, Peter; Crowell, Alexander M.; Ringelberg, Carol; Loros, Jennifer J.; Dunlap, Jay C

    2015-01-01

    Neurospora crassa is an important model organism for filamentous fungi as well as for circadian biology and photobiology. Although the community-accumulated tool set for the molecular analysis of Neurospora is extensive, two components are missing: (1) dependable reference genes whose level of expression are relatively constant across light/dark cycles and as a function of time of day and (2) a catalog of primers specifically designed for real-time PCR (RT-PCR). To address the first of these ...

  19. Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR

    OpenAIRE

    Choi, Hoseong; Cho, Won Kyong; Yu, Jisuk; Lee, Jong-Seung; Kim, Kook-Hyung

    2013-01-01

    To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful prime...

  20. EXPRESSION OF MOUSE Tbx2 GENE IN NORMAL AND MALIGNANTMELANOPHORES BY RT-PCR

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To observe the expression of mouse Tbx2 gene in normal and malignant melanophore. Methods: The normal and malignant cells were used to extract total RNA. The expression of the Tbx2 gene was detected by RT-PCR. Results: No expression of the Tbx2 gene in the normal melanocytes was noted, but all malignant cells showed expression of the Tbx2 gene. Conclusion: Tbx2 plays a critical role during the development of the malignant cells.

  1. Screening of prognostic factors using multiplex RT-PCR technique on different leukemic cell lines

    Directory of Open Access Journals (Sweden)

    Ahani R

    2009-04-01

    Full Text Available "nBackground: Leukemia is one of the most common pediatric malignancies. T-cell Acute Lymphoblastic Leukemia (T-ALL accounts for 15% of hematopoetic cancers. It has been well understood that identification of genetic alterations associated with leukemias is very critical. The molecular genetic techniques have promoted the identification of leukemia-associated genetic changes that may characterize the most accurate predictors of clinical outcome. These considerations reinforce the requirement for rapid identification of the abnormalities. "nMethods: Multiplex RT-PCR, a highly sensitive and specific method applied to screen simultaneously three most frequent transcription factors, TLX1/HOX11, TLX3/HOX11L2 and TAL1/SCL which are associated with T-cell Acute Lymphoblastic Leukemia (T-ALL. "nResults: We describe here our efforts to establish a multiplex RT-PCR analysis system that facilitates the detection of HPB-ALL and K562 cell lines, respectively. "nConclusion: The multiplex RT-PCR technique is a sensitive, valuable and cost-effective diagnostic tool which could improve our ability to accurately and rapidly risk-stratification of patients with childhood T-ALL. In order to perform multiplex RT-PCR technique researchers do not need bone marrow samples and they can employ this method using peripheral blood samples. Therefore, the status of treatment could be followed by assessment of the level of mRNA expression of oncogenic transcriptional factor using peripheral blood sample. Use of this procedure not only provides the best results in short term for specialist, but also clinicians could have opportunities to choose suitable treatment strategies with decrement of drug side effects.

  2. Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues

    OpenAIRE

    Zhang, Fan; Wang, Zhuo-min; Liu, Hong-Yu; Bai, Yun; Sen WEI; Ying LI; Wang, Min; Chen, Jun; Zhou, Qing-hua

    2009-01-01

    Aim: To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method. Methods: Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-ti...

  3. Comparison of hybrid capture and reverse transcriptase polymerase chain reaction methods in terms of diagnosing human cytomegalovirus infection in patients following hematopoietic stem cell transplantation

    International Nuclear Information System (INIS)

    Human cytomegalovirus (CMV) is a life threatening cause of infection among hematopoietic stem cell recipients. Developing reliable methods in detecting the CMV infection is important to identify the patients at risk of CMV infection and disease. The aim of this study was to compare the 2 tests- hybrid capture test, which is routinely used in the diagnosis of CMV infection among hematopoietic stem cell recipients, and reverse transcriptase polymerase chain reaction (RT-PCR) detecting UL21.5 mRNA transcripts of the active virus. In this prospective study, a total of 178 blood samples obtained 35 patients following allogeneic hematopoietic stem cell transplantation at the Bone Marrow Transplantation Unit of the Hematology Department, Ibn-i-Sina Hospital of Ankara University School of Medicine, Turkey between January 2003 and September 2003 were analyzed. Hybrid capture and RT-PCR using UL21.5 gene transcript method to investigate HCMV in blood samples were performed at the department of Microbiology and Clinic Microbiology, Ankara University School of Medicine, Turkey. When Hybrid capture test was accepted as the golden standard, the sensitivity of Rt-PCR was 3%, specificity 100%, false negativity 67%, false positivity 0%, positive predictive value 100%, negative predictive value 74%, and accuracy was 77%. Improving this test by quantification, and application of additional gene transcripts, primarily the late gene transcripts can help increase the sensitivity and feasibility. (author)

  4. Influence of storage time on DNA of Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae for accurate detection by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Lu, Y; Rong, C Z; Zhao, J Y; Lao, X J; Xie, L; Li, S; Qin, X

    2016-01-01

    The shipment and storage conditions of clinical samples pose a major challenge to the detection accuracy of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) when using quantitative real-time polymerase chain reaction (qRT-PCR). The aim of the present study was to explore the influence of storage time at 4°C on the DNA of these pathogens and its effect on their detection by qRT-PCR. CT, NG, and UU positive genital swabs from 70 patients were collected, and DNA of all samples were extracted and divided into eight aliquots. One aliquot was immediately analyzed with qRT-PCR to assess the initial pathogen load, whereas the remaining samples were stored at 4°C and analyzed after 1, 2, 3, 7, 14, 21, and 28 days. No significant differences in CT, NG, and UU DNA loads were observed between baseline (day 0) and the subsequent time points (days 1, 2, 3, 7, 14, 21, and 28) in any of the 70 samples. Although a slight increase in DNA levels was observed at day 28 compared to day 0, paired sample t-test results revealed no significant differences between the mean DNA levels at different time points following storage at 4°C (all P>0.05). Overall, the CT, UU, and NG DNA loads from all genital swab samples were stable at 4°C over a 28-day period. PMID:27580005

  5. Diagnostic accuracy of an IgM enzyme-linked immunosorbent assay and comparison with 2 polymerase chain reactions for early diagnosis of human leptospirosis.

    Science.gov (United States)

    Vanasco, N B; Jacob, P; Landolt, N; Chiani, Y; Schmeling, M F; Cudos, C; Tarabla, H; Lottersberger, J

    2016-04-01

    Enzyme-linked immunosorbent assay (ELISA) tests and polymerase chain reaction (PCR) may play a key role for early detection and treatment of human leptospirosis in developing countries. The aims of this study were to develop and validate an IgM ELISA under field conditions and to compare the diagnostic accuracy among IgG, IgM ELISAs, conventional PCR (cPCR), and real-time PCR (rtPCR) for early detection of human leptospirosis. Overall accuracy of IgM ELISA was sensitivity of 87.9%, specificity of 97.0%, and area under the curve of 0.940. When the 4 methods were compared, IgM ELISA showed the greatest diagnostic accuracy (J=0.6) followed by rtPCR (J=0.4), cPCR (J=0.2) and IgG ELISA (J=0.1). Our results support the use of IgM ELISA and rtPCR for early diagnosis of the disease. Moreover, due to their high specificity, they could be also useful to replace or supplement microscopic agglutination test as a confirmatory test, allowing more confirmations. PMID:26867967

  6. Detection of EWS-FLI1 fusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors by nested reverse transcription polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Qixing Gong; Qinhe Fan; Zhihong Zhang; Weiming Zhang

    2005-01-01

    Objective: To assess the feasibility and significance of detecting EWS-FLIlfusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors (PNETs) by nested reverse transcription polymerase chain reaction (RT-PCR).Methods: Twelve formalin-fixed and paraffin-embedded (FFPE) samples of PNET were retrieved from archive and consultation materials,together with eight cases of controlled tumor. EWS-FLI1 fusion transcripts were detected by nested RT-PCR. Home-keeping gene β-actin was used to detect the quality of mRNA. Results: β-actin mRNA was detected in 9 of the 12 tumor cases. EWS-FLI1 fusion transcripts were detected in 6 cases, among which 4 had a "type 1" fusion transcript and 2 had a "type 2" fusion transcript. None of the controlled tumor was detected the fusion gene. Conclusion: RT-PCR is a feasible method for the detection of EWS-FLI1 fusion transcripts in FFPE tissues in PNET and the result is meaningful in differential diagnosis and prognostic evaluation.

  7. Virus detection and identification using random multiplex (RT-PCR with 3'-locked random primers

    Directory of Open Access Journals (Sweden)

    Eaton John W

    2007-06-01

    Full Text Available Abstract Background PCR-based detection and identification of viruses assumes a known, relatively stable genome. Unfortunately, high mutation rates may lead to extensive changes in viral nucleic acid sequences making dedicated PCR primer use problematic. Furthermore, in bioterrorism, viral consensus sequences can be genetically modified as a countermeasure to RT-PCR and DNA chip detection. Accordingly, there is a great need for the development of rapid and universal virus detection and identification technologies. Results We report herein that viral genomic DNA or RNA can be separated from host nucleic acids in plasma by filtration and nuclease digestion, and randomly amplified in a single PCR using a mixture of primers designed to be resistant to primer-dimer amplification (5'-VVVVVVVVAA-3', V = A, G or C; 38 or 6561 primers. We have termed this novel PCR method Random Multiplex (RT-PCR since hundreds of overlapping PCR amplifications occur simultaneously. Using this method, we have successfullydetected and partially sequenced 3 separate viruses in human plasma without using virus-specific reagents (i.e., Adenovirus Type 17, Coxsackievirus A7, and Respiratory Syncytial Virus B. The method is sensitive to ~1000 genome equivalents/ml and may represent the fastest means of detection of unknown viruses. Conclusion These studies suggest that the further development of random multiplex (RT-PCR may lead to a diagnostic assay that can universally detect viruses in donated blood products as well as in patients suffering with idiopathic disease states of possible viral etiology.

  8. Mass scale screening of common arboviral infections by an affordable, cost effective RT-PCR method

    Institute of Scientific and Technical Information of China (English)

    Debjani Taraphdar; Arindam Sarkar; Shyamalendu Chatterjee

    2012-01-01

    Objective:To develop a rapid, cost effective RT-PCR method for the mass scale diagnosis of such diseases at the viremia stage to find out the actual disease burden in that area. Methods:For this purpose, cases with the history of only short febrile illness were considered. Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome (AES) were selected. Results:Out of 157 samples, 42 and 74 were detected as dengue and chikungunya, respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method. Conclusions:This cost effective RT-PCR method can detect the total positive cases that remain undetected by ELISA method. Moreover, this method is capable to detect the viral RNA from patients’ sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.

  9. Establishment and Application of Real-time Fluorogenetic Quantitative RT-PCR for Detection of Classical Swine Fever Virus%猪瘟病毒实时荧光定量RT-PCR

    Institute of Scientific and Technical Information of China (English)

    李军; 杨威; 潘艳; 禤雄标; 胡帅; 马春霞; 谢宇舟; 陈泽样; 许力干; 谢永平

    2011-01-01

    根据GenBank公布的猪瘟病毒基因组5'非编码区基因序列进行同源性比较分析,选择保守序列区作为扩增区域,设计1对特异性扩增引物,通过优化反应条件,建立了一个用于猪瘟病毒快速定量检测的SY13R GreenⅠ荧光定量RT-PC R方法.试验结果表明该方法重复性好,反应批内循环阈值差异不显著.与猪繁殖与呼吸综合征病毒、伪狂犬病病毒和猪圆环病毒2型等猪源病毒无交叉反应,具有高度的特异性,而且灵敏度高,最小检出量为2×10(2)病毒基因组拷贝数.利用此方法对15例临床样本进行检测,其结果与兔体交又免疫试验一致,表明此方法可作为猪瘟实验室快速诊断和疫情监测的一种快速、准确、简便的检测工具.%To establish a real-time fluorogenetic quantitative RT-PCR assay for detection of classical swine fever virus (CSFV), the 5' non-translated region sequences of CSFV in GenBank were aligned and a pair of specific primers was designed from the conserved sequence within 5' non-translated region.The reactive conditions were optimized to improve the sensitivity and specificity of the assay.The results of reproducibility of this assay were reliable and the intra assay variations were not significant.The specificity test proved that this assay had a high specificity which could not detected PRRSV,PRV and PCV2.The assay also proven to be specific,and the detection limit was up to 2 × 102 copies.The accuracy of real-time fluorogenetic quantitative RT-PCR was evaluated by testing 15 clinical samples.The results of real-time fluorogenetic quantitative RT-PCR were consistent with results of rabbit-cross reaction test,which suggested that real-time quantitative RT-PCR assay may be used as a powerful tool for repaid detection of CSFV.

  10. 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.

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

    Science.gov (United States)

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

    2009-09-10

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

  12. Universal single-probe RT-PCR assay for diagnosis of dengue virus infections.

    Directory of Open Access Journals (Sweden)

    Erik Alm

    2014-12-01

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

  13. Combined Detection of Breast Cancer Micrometastases in the Lymph Nodes and Bone Marrow Using Reverse-transcriptase chain Reaction and Southern Hybridization

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The presence of lymph nodes and bone marrow micrometastases of patients with breast carcinoma by immunohistochemistry (IHC) methods has been strongly correlated to early recurrence and shorter overall survival. The aim of this study was to detect micrometastases in matched sample pairs of lymph nodes and the bone marrow of primary breast cancer patients using a more sensitive method, and compare with other clinical parameters. Methods: Cytokeratin 19 (CK-19) gene mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization. Human breast cancer cell line T47D was mixed with bone marrow cells at different proportions. The positive detection rate was compared among RT-PCR, Southern blotting and IHC methods. Results: Cytokeratin 19 gene was expressed in all 6 positive control samples, while the expression wasn't seen in 18 negative control samples. CK-19 IHC positive cells were detected at a dilution of one T47D cell in 5×105 bone marrow cells, while the sensitivity detected by PCR and Southern blot hybridization was at 1:5′ 104 and 1:106, respectively. In the samples from the 35 patients, we found CK-19 positive cells in 2 cases (5.7%) by IHC. CK-19 gene expression signal was detected in 14/35 (40%) by RT-PCR,and 17/35 (48.6%) by southern blotting. Four cases were micrometastases positive both in lymph node and bone marrow (11.4%). There was no correlation between CK-1 9 detection and other clinical parameters. Conclusion: combined detection of micrometastases in lymph node and bone marrow by RT-PCR and Southern blotting, using CK-19 as a biological marker, is a highly sensitive method for breast cancer.

  14. Classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the European network of excellence for epizootic disease diagnosis and control

    DEFF Research Database (Denmark)

    Hoffmann, Bernd; Blome, Sandra; Bonilauri, Paolo;

    2011-01-01

    The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT......-PCR protocols and a standardized protocol commonly used by the Friedrich-Loeffler-Institute (FLI) on a panel of well-characterized samples. In general, all participants produced results within the acceptable range. The FLI assay, several in-house assays, and the commercial kits had high analytical sensitivity...... and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without...

  15. Development of Reverse Transcription (RT)-PCR and Real-Time RT-PCR Assays for Rapid Detection and Quantification of Viable Yeasts and Molds Contaminating Yogurts and Pasteurized Food Products

    OpenAIRE

    Bleve, Gianluca; Rizzotti, Lucia; Dellaglio, Franco; Torriani, Sandra

    2003-01-01

    Reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays have been used to detect and quantify actin mRNA from yeasts and molds. Universal primers were designed based on the available fungal actin sequences, and by RT-PCR they amplified a specific 353-bp fragment from fungal species involved in food spoilage. From experiments on heat-treated cells, actin mRNA was a good indicator of cell viability: viable cells and cells in a nonculturable state were detected, while no signal was observ...

  16. Diagnosis of bovine foot and mouth disease virus by real-time polymerase chain reaction and nucleotide sequencing from outbreak herd samples in Ilesha Baruba, Kwara state, Nigeria

    Directory of Open Access Journals (Sweden)

    Olatunde Hamza Olabode

    2014-10-01

    Full Text Available Aim: Molecular diagnosis of bovine foot and mouth disease virus (FMDV from outbreak herd in Bukaru-Rontuwa, Sinawu/Tumbunya ward of Ilesha Baruba, in Kwara state-Nigeria was conducted to establish the associated serotypes and disease control plan. Materials and Methods: Purposive study was conducted in cattle outbreak herds during the dry season of January-March, 2011. Random sampling of blood and observed epithelial tissues was collected, stored in accordance with standard methods and subjected to RNA extraction and real-time reverse transcription polymerase chain reaction (rRT-PCR. Positive samples for FMDV were further subjected to reverse transcription polymerase chain reaction (RT-PCR, nucleotide sequencing using sequence primers of serotypes O, A, SAT 1-3 and gel electrophoresis. Obtained data were interpreted based on NCBI BLASTN program. Results: Foot and mouth disease (FMD-RNA extract was not found in all the blood tested with beta-actin range of Ct = 30-34. rRT-PCR assay showed two positive samples with Ct values of 18.79 and 15.28. Gel electrophoresis identified sequenced PCR amplicons as serotype A and SAT 2 respectively. Direct product sequencing confirmed SAT 2 serotype was closely related to SAT 2 isolate LIB/7/2003. Cloned RT-PCR product in pGEM-T easy vector confirmed serotype A as closely related to sequence of A/NIG/21/2009, though multiple NIG/2009 sequences were also identified as closely related. Both isolates showed marked genetic homogeneity with >93% genetic identity in the VP1 region which confirmed heterogeneity and antigenic variation nature of FMDV. Conclusion: Quasi species and subtypes of FMD serotypes A and SAT 2 similar to A/NIG/21/2009 and SAT 2/LIB/7/2003 respectively caused the reported FMD outbreaks in Fulani livestock herds investigated. A combined real-time and optimized RT-PCR protocols that would facilitate effective and timely FMD outbreak control plan based on identified serotypes is thus suggested.

  17. TaqMan探针荧光定量PCR检测花生油中掺入棕榈油的研究%Determination of palm oil adulterated in peanut oil with the TaqMan probe -based RT- PCR method

    Institute of Scientific and Technical Information of China (English)

    周慧; 梁宇斌; 吴苏喜; 李晓明; 裴伟; 杨涛

    2011-01-01

    The method of Real - time fluorescence quantitative polymerase chain reaction ( RT - PCR) with TaqMan fluorescent probe was chosen to fast detect the amount of palm oil mixed in peanut oil. MT3 - B gene of palm was selected as target gene to detect palm oil from peanut oil. The primers of MT3 - B and TaqMan probe were designed, MT3 - B gene reconstructed plasmid was built as absolute quantitative criteria for quantitative RT - PCR to establish standard curve. Peanut oil blended with 1% -40% concentration gradient palm oil was extracted DNA to test palm content by RT - PCR. The result showed that the correlation coefficient (R1) of standard curve with logarithmic linear regression analysis was 0.996. When the adulteration of palm oil in peanut oil reached 5% volume, MT3 - B gene of 17.431 copies per milli-liter of mixed oil could be detected . The method showed good sensitivity, specificity and repeatability.%根据棕榈内源基因MT3 -B设计引物和TaqMan探针,采用基因重组技术构建用于检测棕榈基因MT3 -B的重组质粒作为绝对定量标准品,建立标准曲线,对花生油中掺入棕榈油1% ~40%梯度混合油品提取DNA进行棕榈成分定量检测.结果表明,重组质粒标准品荧光定量标准曲线对数线性回归分析相关系数(R2)为0.996;花生油中掺入棕榈油达到5%时,可检出每亳升混合油品中棕榈MT3 -B基因17.431 copies,检测的重复性和特异性好.

  18. Porcine reproductive and respiratory syndrome virus: Interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods

    DEFF Research Database (Denmark)

    Wernike, Kerstin; Bonilauri, Paolo; Dauber, Malte;

    2012-01-01

    To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American...... commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed...

  19. Diffuse large B-cell lymphoma: sub-classification by massive parallel quantitative RT-PCR.

    Science.gov (United States)

    Xue, Xuemin; Zeng, Naiyan; Gao, Zifen; Du, Ming-Qing

    2015-01-01

    Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity with remarkably variable clinical outcome. Gene expression profiling (GEP) classifies DLBCL into activated B-cell like (ABC), germinal center B-cell like (GCB), and Type-III subtypes, with ABC-DLBCL characterized by a poor prognosis and constitutive NF-κB activation. A major challenge for the application of this cell of origin (COO) classification in routine clinical practice is to establish a robust clinical assay amenable to routine formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies. In this study, we investigated the possibility of COO-classification using FFPE tissue RNA samples by massive parallel quantitative reverse transcription PCR (qRT-PCR). We established a protocol for parallel qRT-PCR using FFPE RNA samples with the Fluidigm BioMark HD system, and quantified the expression of the COO classifier genes and the NF-κB targeted-genes that characterize ABC-DLBCL in 143 cases of DLBCL. We also trained and validated a series of basic machine-learning classifiers and their derived meta classifiers, and identified SimpleLogistic as the top classifier that gave excellent performance across various GEP data sets derived from fresh-frozen or FFPE tissues by different microarray platforms. Finally, we applied SimpleLogistic to our data set generated by qRT-PCR, and the ABC and GCB-DLBCL assigned showed the respective characteristics in their clinical outcome and NF-κB target gene expression. The methodology established in this study provides a robust approach for DLBCL sub-classification using routine FFPE diagnostic biopsies in a routine clinical setting. PMID:25418578

  20. Outcome on EPIZONE Extension on VER/ VNN: Diagnostics, proficiency test and qRT-PCR validation

    OpenAIRE

    Bigarré, Laurent; Panzarin, Valentina; Baud, Marine; Jonstrup, Søren Peter; Jansson, Eva; Isaksson, Mats; Engelsma, Marc; Olesen, Niels Jørgen; Bovo, Giuseppe

    2012-01-01

    IntroductionBetanodaviruses are genetically very diverse with at least five genogroups described and a large range of variants within each group, including reassortants carrying components of two groups (Panzarin et al., 2011; Toffolo et al., 2007). Their genome is composed of two strands of RNA: RNA1 and RNA2. Till now, only one universal real-time RT-PCR method has been published which detects all variants of RNA2 (Panzarin et al., 2010) in clinical cases. An additional method based on dete...

  1. Rapid subtyping of tick-borne encephalitis virus isolates using multiplex RT-PCR

    Czech Academy of Sciences Publication Activity Database

    Růžek, Daniel; Šťastná, B.; Kopecký, Jan; Golovljova, I.; Grubhoffer, Libor

    2007-01-01

    Roč. 144, 1/2 (2007), s. 133-137. ISSN 0166-0934 R&D Projects: GA MŠk(CZ) LC06009; GA ČR(CZ) GA524/06/1479 Grant ostatní: Grantová agentura Jihočeské univerzity(CZ) 44/2006/P-BF; Estonian Science Foundation(EE) 5963; GAMŠk(CZ) FRVŠ 230/2007 Institutional research plan: CEZ:AV0Z60220518 Keywords : tick-borne encephalitis virus * subtyping * multiplex RT-PCR Subject RIV: EE - Microbiology, Virology Impact factor: 1.933, year: 2007

  2. Presumptive detection of yellow head virus by reverse transcriptase-polymerase chain reaction and dot-blot hybridization in Litopenaeus vannamei and L. stylirostris cultured on the Northwest coast of Mexico.

    Science.gov (United States)

    de la Rosa-Vélez, J; Cedano-Thomas, Y; Cid-Becerra, J; Méndez-Payán, J C; Vega-Pérez, C; Zambrano-García, J; Bonami, J-R

    2006-12-01

    In order to assess the presence of yellow head virus (YHV) in shrimp farms along the Pacific coast of Mexico, 39 samples from 26 randomly chosen farms were analysed by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridization. Eleven samples were positive for YHV. The disease was reproduced by means of an infectivity bioassay performed with an extract of pleopods from the positive samples. Cumulative mortality reached 50% in 14 days. Four pairs of primers which amplified several YHV genome regions were designed and used to test dead and surviving shrimp from the bioassay by RT-PCR, resulting in positive results for every expected amplicon. The results of this study provide presumptive evidence of the presence of YHV in Mexican shrimp farms at least during 1999-2000. PMID:17169104

  3. Selection of Housekeeping Genes for Transgene Expression Analysis in Eucommia ulmoides Oliver Using Real-Time RT-PCR

    OpenAIRE

    Yoshihisa Nakazawa; Koichiro Gyokusen; Ren Chen; Mayumi Gyokusen

    2010-01-01

    In order to select appropriate housekeeping genes for accurate calibration of experimental variations in real-time (RT-) PCR results in transgene expression analysis, particularly with respect to the influence of transgene on stability of endogenous housekeeping gene expression in transgenic plants, we outline a reliable strategy to identify the optimal housekeeping genes from a set of candidates by combining statistical analyses of their (RT-) PCR amplification efficiency, gene expression st...

  4. Detection of canine distemper virus nucleoprotein gene by RT-PCR in urine of dogs with distemper clinical signs

    International Nuclear Information System (INIS)

    The urine of 87 dogs with clinical signs suggestive of canine distemper was analyzed by RT-PCR for detection of canine distemper virus (CDV) nucleoprotein gene. The samples were allotted to the following groups: group A- with 41 dogs with systemic symptoms, group B- with 37 dogs with neurological signs, and group C- with 9 dogs with simultaneous systemic and neurological clinical signs. Group D (control) included 20 assymptomatic dogs. A c2 was used to test RT-PCR results according to clinical form and hematological characteristics. The RT-PCR was positive for CDV in 47% (41/87) of the urine samples from dogs with clinical signs. All samples from assymptomatic dogs were RT-PCR negatives. Positive samples were found in all groups of dogs with distemper symptoms according to the following propositions: 51.2% (21/41), 29% (11/37) and 100% (9/9) for groups A, B and C, respectively. In all clinical forms (groups A, B and C) leucocytosis was the most frequent observed hematological alteration. No relationship between RT-PCR results and hematological changes was observed. The results showed that independently of the clinical stage of the illness the RT-PCR based on urine sample can be applied for ante mortem diagnosis of CDV

  5. Nested RT-PCR for ante mortem diagnosis of rabies from body secretion/excretion of animals suspected for rabies

    Directory of Open Access Journals (Sweden)

    M Dandale

    Full Text Available Aim: The present study deals with molecular technique Nested RT-PCR for detection of rabies viral RNA from biological fluid samples (Saliva, Milk and Urine collected from animal suspected for rabies and to compare the sensitivity of Nested RT-PCR applied for ante mortem diagnosis of rabies with conventional technique (immunofluorescence applied on neural tissue. Materials and Methods: Nested RT-PCR was applied on 62 biological fluid specimens collected from rabies suspected animals. First round amplification with nested set of primers (RabN1 and RabN5 yielded 1477 bp product while amplification with second round primers (RabNfor and RabNrev yielded 762 bp product. Sensitivity of the technique was compared in accordance with WHO recommended gold standard test viz. Immunofluorescence (FAT applied on brain samples. Results: By Nested RT-PCR, viral RNA could be detected in 9/24 (37.50% saliva samples, 2/17 (11.76% milk samples and 6/21 (28.57% urine samples. Confirmatory diagnosis by Immunofluorescence performed on brain sample revealed 18 true positive cases. Overall, Sensitivity of Nested RT-PCR technique employed on fluid samples was 69.23% when compared with immunofluorescence performed on brain samples. Conclusions: Early reliable ante mortem diagnosis of rabies can be obtained from biological fluid samples of animals suspected to be rabid when tested with Nested RT-PCR technique. [Vet World 2012; 5(11.000: 690-693

  6. Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer

    Directory of Open Access Journals (Sweden)

    Coppola Domenico

    2006-10-01

    Full Text Available Abstract Background The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. Methods In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22 that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR. Results Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. Conclusion The design of new approaches to identify such markers is warranted.

  7. Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) phthalate using cDNA microarray and real-time RT-PCR

    International Nuclear Information System (INIS)

    To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment

  8. Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen.

    Science.gov (United States)

    Pérez-Rico, A; Crespo, F; Sanmartín, M L; De Santiago, A; Vega-Pla, J L

    2014-10-01

    Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thawing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate amplification, specificity, and stability to be used as future reference genes in expression studies. Live spermatozoa were selected from cryopreserved semen straws derived from 20 stallions, through a discontinuous concentration gradient. RNA purification followed a combination of the organic and column extraction methods together with a deoxyribonuclease treatment. The selective amplification of nine candidate genes was undertaken using reverse transcription and real-time polymerase chain reaction (qPCR) carried out in a one-step mode (qRT-PCR). Specificities were tested by melting curves, agarose gel electrophoresis and sequencing. In addition, gene stabilities were also calculated. Results indicated that five out of the nine candidate genes amplified properly (β-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein and Ubiquitin B), of which β-Actin and the L32 Ribosomal protein showed the highest stability thus being the most suitable to be considered as reference genes for equine cryopreserved sperm studies, followed by the ATP synthase subunit beta and Ubiquitin B. PMID:25192831

  9. Evaluation of RT-PCR to Detect Translocations in Children Diagnosed with Acute Lymphoblastic Leukemia

    Directory of Open Access Journals (Sweden)

    H Galehdari

    2009-12-01

    Full Text Available "nBackground: Acute lymphoblastic leukemia (ALL is the most common subtype of childhood cancer. Chromosomal ab­normality, specially the replacement of chromosomal material is one of the main reasons in generating leukemia, wherein the kind of translocation play a key role in managing the remedy. The goal of the present study was to develop a reliable, rapid, and cost effective method to detect translocations, which are the main sources of leukemia."nMethods: Twenty seven samples were collected from leukemia affected individuals that were referred to the Shafa Hospital in Ahwaz from summer 2007 to spring2008. Total RNA was extracted from one milliliter whole blood, and then reversely tran­scribed using reverse transcriptase. Finally, multiplex RT-PCR was performed for each sample."nResults: Cell lines (K562, Jurkat E 6.1 that are harboring known translocations were used as positive control, with addi­tional internal control to prove false negative results. Translocations t (9; 22, t (12; 21, t (1; 19 and t (4; 11 were ob­served in patients that have been diagnosed with the ALL, respectively. No Translocation has been seen in individuals suf­fering lymphoma."nConclusion: Multiplex RT-PCR assay is an effective, sensitive, accurate, and cost-effective diagnostic tool, which can im­prove the ability to accurately and rapidly risk-stratify patients that were diagnosed with acute lymphoblastic leukemia.

  10. Evaluation of gamma-sterilization (60Co) by RT-PCR by DHFR expression detection

    International Nuclear Information System (INIS)

    The improvement of techniques to detect pathogen agents in blood had reduced significantly the contamination mechanisms by hemocomponents in blood transfusion procedures. Ionizing radiation is a method that has presented several applications on medicine and in currently days has been showing special attention on blood banks which has been applied to avoid TA-GVHD development. DHFR is an enzyme constitutive in Plasmodium protozoa and has an important role in folate metabolism on these parasites. Detecting the expression of RNAm coder for this enzyme is possible to evaluate the viability of this parasite in blood samples. Plasmodium chabaudi AJ is a parasite that induces lethal malaria in rodents similar to human malaria In this work, the objective was to detect the presence of plasmodium protozoa in irradiated blood samples, infected experimentally, through the application of a RT-PCR using primers for the coder sequence of DHFR's mRNA. We studied doses of ionizing radiation between 0 and 75 Gy. The irradiation procedures were accomplished in Center of Radiation Technology of IPEN-CNEN in a 60Co panoramic source. Our results had demonstrated that RT-PCR is a sensible method to evaluate the viability of plasmodium in blood samples because the technique could detect low parasite burden in all tested samples. (author)

  11. Evaluation of gamma-sterilization ({sup 60}Co) by RT-PCR by DHFR expression detection

    Energy Technology Data Exchange (ETDEWEB)

    Converso, Ana Paula G.; Andrade Junior, Heitor F. de [Instituto de Medicina Tropical de Sao Paulo, Sao Paulo, SP (Brazil)]. E-mail: anapaulagconverso@gmail.com; hfandrad@usp.br; Vieira, Daniel P. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Instituto de Medicina Tropical de Sao Paulo, Sao Paulo, SP (Brazil); E-mail: dperezv@usp.br

    2007-07-01

    The improvement of techniques to detect pathogen agents in blood had reduced significantly the contamination mechanisms by hemocomponents in blood transfusion procedures. Ionizing radiation is a method that has presented several applications on medicine and in currently days has been showing special attention on blood banks which has been applied to avoid TA-GVHD development. DHFR is an enzyme constitutive in Plasmodium protozoa and has an important role in folate metabolism on these parasites. Detecting the expression of RNAm coder for this enzyme is possible to evaluate the viability of this parasite in blood samples. Plasmodium chabaudi AJ is a parasite that induces lethal malaria in rodents similar to human malaria In this work, the objective was to detect the presence of plasmodium protozoa in irradiated blood samples, infected experimentally, through the application of a RT-PCR using primers for the coder sequence of DHFR's mRNA. We studied doses of ionizing radiation between 0 and 75 Gy. The irradiation procedures were accomplished in Center of Radiation Technology of IPEN-CNEN in a {sup 60}Co panoramic source. Our results had demonstrated that RT-PCR is a sensible method to evaluate the viability of plasmodium in blood samples because the technique could detect low parasite burden in all tested samples. (author)

  12. A simple RT-PCR-based strategy for screening connexin identity

    Directory of Open Access Journals (Sweden)

    M. Urban

    1999-08-01

    Full Text Available Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

  13. Strategy for the maximization of clinically relevant information from hepatitis C virus, RT-PCR quantification.

    LENUS (Irish Health Repository)

    Levis, J

    2012-02-03

    BACKGROUND: The increasing clinical application of viral load assays for monitoring viral infections has been an incentive for the development of standardized tests for the hepatitis C virus. OBJECTIVE: To develop a simple model for the prediction of baseline viral load in individuals infected with the hepatitis C virus. METHODOLOGY: Viral load quantification of each patient\\'s first sample was assessed by RT-PCR-ELISA using the Roche MONITOR assay in triplicate. Genotype of the infecting virus was identified by reverse line probe hybridization, using amplicons resulting from the qualitative HCV Roche AMPLICOR assay. RESULTS: Retrospective evaluation of first quantitative values suggested that 82.4% (n=168\\/204) of individuals had a viral load between 4.3 and 6.7 log(10) viral copies per ml. A few patients (3.4%; n=7\\/204) have a serum viremia less than the lower limit of the linear range of the RT-PCR assay. Subsequent, prospective evaluation of hepatitis C viral load of all new patients using a model based on the dynamic range of viral load in the retrospective group correctly predicted the dynamic range in 75.9% (n=33\\/54). CONCLUSION: The dynamic range of hepatitis C viremia extends beyond the linear range of the Roche MONITOR assay. Accurate determination of serum viremia is substantially improved by dilution of specimens prior to quantification.

  14. A quantitative real time polymerase chain reaction approach for estimating processed animal proteins in feed: preliminary data

    Directory of Open Access Journals (Sweden)

    Maria Cesarina Abete

    2013-04-01

    Full Text Available Lifting of the ban on the use of processed animal proteins (PAPs from non-ruminants in non-ruminant feed is in the wind, avoiding intraspecies recycling. Discrimination of species will be performed through polymerase chain reaction (PCR, which is at a moment a merely qualitative method. Nevertheless, quantification of PAPs in feed is needed. The aim of this study was to approach the quantitative determination of PAPs in feed through Real Time (RT-PCR technique; three different protocols picked up from the literature were tested. Three different kind of matrices were examined: pure animal meals (bovine, chicken and pork; one feed sample certified by the European reference laboratory on animal proteins (EURL AP in feed spiked with 0.1% bovine meal; and genomic DNAs from bovine, chicken and pork muscles. The limit of detection (LOD of the three protocols was set up. All the results obtained from the three protocols considered failed in the quantification process, most likely due to the uncertain copy numbers of the analytical targets chosen. This preliminary study will allow us to address further investigations, with the purpose of developing a RT-PCR quantitative method.

  15. RT-PCR em pools de soros sangüíneos para o diagnóstico da infecção aguda e de animais persistentemente infectados pelo vírus da diarréia viral bovina RT-PCR in pools of bovine blood serum to detect acute infection and persistently infected animals with bovine viral diarrhea virus

    Directory of Open Access Journals (Sweden)

    D. Pilz

    2007-02-01

    Full Text Available Utilizou-se a técnica da RT-PCR para a detecção da região 5' UTR do genoma do vírus da diarréia viral bovina (BVDV em pools de soros sangüíneos provenientes de um rebanho, constituído por 226 animais, que apresentava distúrbios da reprodução. A partir das amostras individuais de soro e de acordo com a categoria dos animais e o número de animais por categoria foram formados 10 pools (A a J de soros. A primeira avaliação revelou a amplificação de um produto com 290pb nas reações referentes aos grupos D (35 vacas e H (25 bezerros lactentes que, após o desmembramento em amostras individuais, resultou na identificação de 11 vacas lactantes e 12 bezerros em amamentação positivos. Para a identificação de animais persistentemente infectados (PI entre os 23 positivos na primeira avaliação, realizou-se a segunda colheita de soros sangüíneos, três meses após. A RT-PCR das amostras individuais de soro revelou resultado positivo em cinco bezerros. Em dois, foi possível isolar o BVDV em cultivo de células MDBK. A especificidade das reações da RT-PCR foi confirmada pelo seqüenciamento dos produtos amplificados a partir do soro de uma vaca com infecção aguda, de um bezerro PI e das duas amostras do BVDV isoladas em cultivo celular. A utilização da RT-PCR em pools de soros sangüíneos demonstrou ser uma estratégia rápida de diagnóstico etiológico e de baixo custo tanto para a detecção de infecção aguda quanto de animais PI.The 5' untranslated region of the bovine viral diarrhea virus (BVDV genome was detected by RT-PCR assay in pools of blood sera samples collected from a cattle herd (n=226 animals with reproductive failures. Based on the classes of animal and the number of animals per class, the individual blood serum samples were distributed in 10 sera pools (A to J. During the first evaluation a 290bp amplicon was amplified in reactions from groups D (35 cows and H (25 sucking calves. The individual analysis

  16. Polymerization as a Model Chain Reaction

    Science.gov (United States)

    Morton, Maurice

    1973-01-01

    Describes the features of the free radical, anionic, and cationic mechanisms of chain addition polymerization. Indicates that the nature of chain reactions can be best taught through the study of macromolecules. (CC)

  17. 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

    Directory of Open Access Journals (Sweden)

    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

  18. Free cancer cell detection in peritoneal cavity in gastric cancer patients by RT-PCR for CEA

    International Nuclear Information System (INIS)

    Authors applied RT-PCR assay to detecting CEA expressing free cancer cells in peritoneal cavity of 114 gastric cancer patients to find an indication for prophylactic treatment to prevent peritoneal recurrence. Sixty-three of 114 cases were positive for RT-PCR, of which 16 cases were positive for cytologic examination and 47 cases were negative. Forty-nine of 51 cases who were negative for RT-PCR were negative for cytologic examination. Positivity for RT-PCR according to the depth of invasion were as follows : two (28.6 %) of seven cases whose cancer invaded mucosal or submucosal layer were positive. Ten (45.5 %) of 22 cases whose cancer invaded muscular or subserosal layer were positive. Forty-one (57.7 %) of 71 serosa involved cases were positive. Eleven (78.6 %) of cases who had grossly perioneal seedings were positive (p=0.026). However, all of 7 EGC cases, 19 of 22 cases whose cancer invaded to muscle layer or to subserosa were negative for cytologic examination, and eight of 13 cases who had had peritoneal seedings were positive. Positivity for RT-PCR according to cell differentiation were as follows: forty-two (61.8 %) of 68 cases who cancer were poorly differentiated type were positive. (p=0.163) Serum level of CEA of RT-PCR positive group and that of negative group were not statistically different. It was revealed that RT-PCR was more sensitive than cytologic examination in detecting free tumor cells, especially in pm, ss and serosa positive cancers, so if further study with more cases and longer follow-up is performed, its role as prognostic factor and an indication of prophylactic therapy will be clarified. (author). 22 refs., 5 tabs

  19. [Development of a real-time polymerase chain reaction method for the identification of Candida species].

    Science.gov (United States)

    Ağca, Harun; Dalyan Cilo, Burcu; Özmerdiven, Gülşah Ece; Sağlam, Sezcan; Ener, Beyza

    2015-01-01

    Candida species are one of the major causes of nosocomial infections and are the fourth most common agent involved in bloodstream infections. The impact of non-albicans Candida species is increasing, however C.albicans is still the most common species. Since the antifungal susceptibility pattern among Candida spp. may be different, rapid diagnosis and identification of non-albicans Candida spp. are important for the determination of antifungal agents that will be used for treatment. The aim of the study was to describe a real-time polymerase chain reaction (Rt-PCR) assay that rapidly detects, identifies and quantitates Candida species from blood culture samples. A total of 50 consecutive positive blood culture bottles (BACTEC, Beckton Dickinson, USA) identified at our laboratory between June-November 2013, were included in the study. Reference strains of Candida spp. (C.albicans ATCC 10231, C.glabrata ATCC 90030, C.tropicalis ATCC 1021, C.krusei ATCC 6258, C.parapsilosis ATCC 22019 and C. dubliniensis CD36) grown on Sabouraud dextrose agar were used for quality control. BACTEC bottles that were positive for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were also studied to search the cross-reactivity. A commercial kit (Zymo Research, USA) was used for DNA extraction. Real-time PCR was performed on LightCycler 480 (Roche, Germany) with primers and probes specific for 18S rRNA of Candida species. Twenty microlitres of the reaction mix contained 2 μl of extracted DNA, 2 μl of LightCycler Fast Start DNA Master Probe (Roche Diagnostics, Germany), 2 μl of MgCl(2) (5 mmol), 2 μl of 10x PCR buffer (Roche Diagnostics, Germany), 0.5 μl of each primer (0.01 nmol/μl) and 1 μl of each probe (0.1 μmol/μl) (TibMolBiol, Germany). Amplification was performed using the following conditions; 95°C for 10 mins and 50 cycles of denaturation at 95°C for 10 secs, annealing at 62°C for 10 secs and polymerisation at 72°C for 20 secs. A melting curve was

  20. Relative quantitative RT-PCR to study the expression of plant nutrient transporters in arbuscular mycorrhizas

    DEFF Research Database (Denmark)

    Burleigh, S.H.

    2001-01-01

    The influence of arbuscular mycorrhizal fungi (AMF) on the expression of plant nutrient transporters was studied using a relative. quantitative reverse-transcription polymerase chain-reaction (RQRT-PCR) technique. Reverse-transcribed 18S rRNA was used to standardize the treatments. The technique...

  1. Viral elution and concentration method for detection of influenza A viruses in mud by real-time RT-PCR.

    Science.gov (United States)

    Deboosere, Nathalie; Horm, Srey Viseth; Delobel, Alexandre; Gachet, Jessica; Buchy, Philippe; Vialette, Michèle

    2012-01-01

    The role of environmental reservoirs in avian influenza virus (AIV) transmission has been investigated during AIV-associated outbreaks. To date, no method has been defined for detection of AIV from mud samples. A procedure using elution and polyethylene glycol (PEG) concentration steps was designed to detect AIV by RT-PCR from 42g of raw mud, corresponding to 30g of the solid fraction of mud. RNA was recovered with MagMAX AI/ND Viral RNA Isolation kit (Ambion, Austin, TX). Three elution buffers were studied and viral recoveries higher than 29% were yielded by elution with a 10% beef extract solution (pH 7). The overall method showed that, under some conditions, virus was not detectable in PEG samples, whereas viruses were detected in the elution fractions. PCR curves were improved significantly by running the amplification reaction with a mixture containing a PCR additive for inhibitor removal, such as T4 gene 32 protein (Gp32), although PCR inhibitors from mud were removed partially from PEG samples. A theoretical detection threshold of 5×10(5) RNA copies of H5N1 virus per 30g of solid mud could be obtained by elution. The overall method has proved successful for detecting H5N1 virus contamination of mud specimens collected during outbreak investigations of avian influenza in Cambodia. PMID:22036660

  2. Utility of Pooled HIV RNA RT-PCR Assay in Diagnosing Acute HIV Infections

    Institute of Scientific and Technical Information of China (English)

    张麒; 蒋岩; 刘全忠

    2004-01-01

    Abstract: The P24 antigen test, HIV RNA PCR test,HIV isolation/culture and fourth-generation HIV uniform Ag/Ab assay are being utilized in diagnosing acute HIV infection in different labs. Many factors limit the use of screening for acute HIV in high-risk populations, in blood donors and during voluntary HIV testing, including, cost, technique, sensitivity and specificity. In this review we explore a new NAAT method which involves HIV RNA RT-PCR on pooled samples. This technique is able to screen for acute infections in a large testing volume and may he used as a screening method in high-risk populations and blood donors.

  3. Evaluation of Microarray Preprocessing Algorithms Based on Concordance with RT-PCR in Clinical Samples

    DEFF Research Database (Denmark)

    Hansen, Kasper Lage; Szallasi, Zoltan Imre; Eklund, Aron Charles; Gyorffy, B; Molnar, B

    2009-01-01

    Several preprocessing algorithms for Affymetrix gene expression microarrays have been developed, and their performance on spike-in data sets has been evaluated previously. However, a comprehensive comparison of preprocessing algorithms on samples taken under research conditions has not been...... performed. METHODOLOGY/PRINCIPAL FINDINGS: We used TaqMan RT-PCR arrays as a reference to evaluate the accuracy of expression values from Affymetrix microarrays in two experimental data sets: one comprising 84 genes in 36 colon biopsies, and the other comprising 75 genes in 29 cancer cell lines. We...... evaluated consistency using the Pearson correlation between measurements obtained on the two platforms. Also, we introduce the log-ratio discrepancy as a more relevant measure of discordance between gene expression platforms. Of nine preprocessing algorithms tested, PLIER+16 produced expression values that...

  4. Detection of enteroviruses and hepatitis a virus RNA in cow milk by RT-PCR

    Directory of Open Access Journals (Sweden)

    Terzi Goknur

    2010-01-01

    Full Text Available The aim of the study is to evaluate the precence of Hepatitis A virus (HAV and enteroviruses (EV in milk samples. Raw milk and whey samples (n=50, collected from eight different open air markets in Samsun (Turkey were analysed for HAV and enteroviruses by RT-PCR. Viral RNA was extracted using the rapid acid guanidinium thiocyanatephenol- chloroform method. HAV primers were selected from sequences of the VP2 and VP4 capsid region. Enterovirus primers were selected from the 5' non-translated region, which is the most conserved region in an enterovirus genome. Four samples (8% raw milk were positive for EV RNA. However, HAV RNA was not determined the milk samples. This is the first report on the detection of HAV and EV RNA in cow milk samples in Turkey. This study highlights the interest for contamination by HAV and EV in milk samples from different areas.

  5. 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 the microbiological identification with the two techniques: Examining culture and Real-Time PCR of Commerce ( Gum-Sunstar that identifies 4 bacterial species: A. actinomycetemcomitans (A.a., P.gingivalis (P.g., T.forsythensis (T.f., and T.denticola (T.d.. Results. All patients studied were positive to both tests with charger high: the consideration of tenure, with CFU / ml > 105, was positive in 66.6% of samples by:T.f., and P.g., in 12.5% for A.a., while T.d. not been sought by examining culture, the RT-PCR was positive, with high loads, in 95.8% of samples for T.f., in 79.1% for P.g., in 12.5% for A.a. and 20.8% for T.d.The test crop showed the presence of even P.intermedia in 91

  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. QUANTIFICATION OF P4HA2 mRNA OF FIBROBLASTS WITH SYBR GREEN BASED RT-PCR FOR CORRECTING CMV INACTIVATION EFFICIENCY IN DONOR BLOOD

    Institute of Scientific and Technical Information of China (English)

    FANG Feng-qin; ZHANG Yue; LU Ping; ZHANG Li; JI Yu-hua

    2009-01-01

    Objective To quantify proline 4-hydroxylase, alpha polypeptide Ⅱ (P4HA2) mRNA of human embryo lung fibroblast (HELF) with SYBR green based reversed transcript PCR (RT-PCR) for correcting cytomegalovirus (CMV) inactivation or clearance efficiency in donor blood.Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated.Results The sensitivity of the method was 1.5E+04 copies/mL of P4HA2 mRNA, corresponding to 103 fibroblasts. In addition, existence of 8.67E+06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13.76% in variation, which showed acceptable stability of this method.Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.

  8. TaqMan探针实时荧光定量反转录-聚合酶链反应检测新布尼亚病毒L基因方法的建立与应用%Establishment of real time RT-PCR assay for detecting novel Bunyavirus L gene

    Institute of Scientific and Technical Information of China (English)

    虞吉寅; 王忠发; 任宜; 毛彬

    2013-01-01

    Objective To develop a sensitive and specific real time reverse transcription PCR ( RT-PCR) assay for nucleic acid detection of novel Bunyavirus. Methods Primers and TaqMan probe were designed according to the L gene of novel Bunyavirus and the real time RT-PCR was used to detect novel Bunyavirus from serum and plasma samples of 50 clinical suspected patients and from serum of 25 healthy people. Two virus strains isolated from the patients' serum were used to evaluate the specificity and sensitivity as well as the reproducibility of the established real time RT-PCR assay. Results The established real time RT-PCR assay had high specificity, with the Ct values showing good linear relation to the copy numbers of template DNA in the standard curve (R2 =0.999). In this assay, the target gene could be detected if the template DNA is more than 25 copies in 25 ΜL PCR reaction, indicating that the sensitive detection limit was 1 copy/μl. The reproducibility tests indicated that the intraassay coefficient of variation were all lower than 1. 0% . Among the 50 patients' serum samples, 30 were positive, 25 control' s serum samples and 28 rats lung and testes samples were all negative. Two of 8 blood samples were positive in virus culture. Conclusion The established real time RT-PCR assay was with high specificity, sensitivity and reproducibility, which could be used in the rapid nucleic acid detection of novel Bunyavirus in humans and animals and applied in the preliminary identification of the virus after isolation and culture.%目的 建立敏感、特异、实时荧光定量反转录-聚合酶链反应(real time fluorescent quantitative reverse transcription-polymerase chain reaction,rRT-PCR)方法,用于新布尼亚病毒的核酸检测.方法 以新布尼亚病毒L基因为靶基因设计引物以及TaqMan探针,建立real-time RT-PCR方法,并对舟山市岱山县50份临床疑似患者血清、血浆、25份正常人群血清和从患者血清中分离到的2株病毒

  9. Nested RT-PCR method for the detection of European avian-like H1 swine inlfuenza A virus

    Institute of Scientific and Technical Information of China (English)

    WEI Yan-di; PEI Xing-yao; ZHANG Yuan; YU Chen-fang; SUN Hong-lei; LIU Jin-hua; PU Juan

    2016-01-01

    Swine inlfuenza A virus (swine IAV) circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 inlfuenza pandemic. Among multiple subtypes/lineages of swine inlfuenza A viruses, European avian-like (EA) H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and speciifc methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription (RT)-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets (outer and inner) were designed speciifcaly to target the viral hemagglutinin genes. Speciifc PCR products were obtained from al tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit (PFU) mL–1which was over 104 PFU mL–1 for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveilance of EA H1 swine IAVs in pigs and humans.

  10. K-19 mRNA RT-PCR in detecting micrometastasis in regional lymph nodes of gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Jian Suo; Quan Wang; Hong-Juan Jin; Hong Li; Hang Zhao

    2006-01-01

    AIM: To investigate the value and prospect of RT-PCR in detecting micrometastasis in regional lymph nodes of gastric cancer.METHODS: Histopathology was used and K19 mRNA expression was detected by RT-PCR in tumor tissues and lymph nodes from gastric cancer patients undergoing radical resection of gastric carcinoma. RESULTS: K19 mRNA was expressed in all tumor specimens of 30 cases; of the 126 lymph nodes, 26 were histopathologically positive (20.6%), and 42 positive (33.3%) by RT-PCR. Amplification fragments of 460 and 540 bp were shown in all the tumor tissues and metastatic lymph nodes after K19 and 3-actin RT-PCR, while only a 540 bp fragment appeared in the lymph nodes of non-tumor patients.CONCLUSION: K19 mRNA RT-PCR is sensitive and specific in testing micrometastasis in regional lymph nodes of gastric cancer, and it is superior to routine histopathology.

  11. Relative efficiency of polymerase chain reaction and enzyme-linked immunosorbant assay in determination of viral etiology in congenital cataract in infants

    Directory of Open Access Journals (Sweden)

    Shyamala G

    2008-01-01

    Full Text Available Background: Perinatal viral infections of fetus are among the leading causes of congenital cataract and identifying the viral etiology is important. Objectives: To detect the presence of Rubella virus (RV, herpes simplex virus (HSV and cytomegalovirus (CMV in lens aspirate specimens obtained from patients with congenital cataract and relate the results with serology. Setting and Design: Prospective study carried out in tertiary care hospital. Materials and Methods: Fifty lens aspirates from 50 infants with congenital cataract were subjected to HSV, RV isolation and polymerase chain reaction (PCR for detection of HSV and CMV. Reverse transcription polymerase chain reaction (RT-PCR was applied for RV detection. Peripheral blood specimens were screened for anti-HSV, RV and CMV antibodies by enzyme-linked immunosorbant assay (ELISA. Results: Rubella virus was detected in nine (18% lens aspirates, by nRT-PCR which includes six positive by culture. HSV-2 DNA was detected in nine other lens aspirates, while CMV was not detected by PCR. Serological results did not correlate with the presence of viruses in the lens aspirates. This is the first report of detection of HSV-2 DNA in cases of congenital cataract. Conclusions: Cytomegalovirus may not be playing a significant role in causation of congenital cataract. The role of serology in identifying causative viral infection for congenital cataract needs to be re-evaluated.

  12. 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.

  13. Triplex-RT-PCR Detection of PVY, PVS and PLRV in Potato%马铃薯上PVY、PVS和PLRV的三重RT-PCR检测

    Institute of Scientific and Technical Information of China (English)

    张华鹏; 张剑峰; 刘俊莹; 王聪聪

    2011-01-01

    根据PVY、PVS和PLRV外壳蛋白(Coat protein,CP)基因序列的保守区域设计各自的引物,利用三重RTPCR技术实现了在同一反应体系中同时扩增出3种病毒产物.PVY、PVS和PLRV扩增产物测序长度均与目的片段的长度相符,分别为781,181,364 bp;各种病毒产物的测序结果同GeneBank中的序列比对后的同源性均高达95%以上.将3种病毒的RNA稀释后进行RT-PCR,PVY、PVS和PLRV的最低检测含量分别为4.5 pg/μL~4.5 fg/μL、3.7fg/μL和4.6 fg/μL.三重RT-PCR为检测单独或复合感染PVY、PVS和PLRV的马铃薯材料,提供了一种方便、高效的分子学方法.%Three pairs of primers for PVY, PVS and PLRV, by comparing and finding out each conserved domain of CP sequence were designed, and then triplex-RT-PCR technology was used to amplify three product fragments of PVY,PVS and PLRV in one reaction simultaneously. The length of the PVY.PVS and PLRV are fit to the length of target DNA,781 ,181 ,364 bp respectively. The homology of the three viruses all reach up to 95% by comparing sequencing result with each published sequences in GeneBank respectively. After diluting the RNA samples, the minimum detection content for PVY,PVS and PLRV is 4. 5 pg/μL -4. 5 fg/μL,3. 7 fg/|iX and 4. 6 fg/μL in RT-PCR respectively. Triplex-RT-PCR technology provides one convenient and high-efficiency molecular method to detect potato materials infected with one or more viruses among PVY,PVS and PLRV.

  14. Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue

    DEFF Research Database (Denmark)

    Green, Tina Marie; de Stricker, Karin; Møller, Michael Boe

    2009-01-01

    paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes and......Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue...... was 0.93 (P<0.01) after normalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes....

  15. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. 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.

  18. 牛病毒性腹泻病毒、牛呼吸道合胞体病毒和牛副流感病毒3型三重RT-PCR检测方法的建立及应用%Establishment and application of a multiple RT-PCR for BVDV, BRSV and BPIV-3

    Institute of Scientific and Technical Information of China (English)

    索阳; 陈继勇

    2013-01-01

    To identify and differentiate rapidly the causing pathogens of clinical diseases,a multiple RT-PCR was developed.The polymerase chain reaction was optimized to simultaneously detect three pathogens,including bovine viral diarrhea virus (BVDV),bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus type 3 (BPIV-3).Three sets of specific primers were designed according to the sequences of BVDV,BRSV and BPIV-3 at the GenBank.Using three pairs of specific primers,a RT-PCR was established to amplify the conservative regions of the three viruses,respectively.The RT-PCR system would amplify a 466 bp fragment for BVDV,a 735 bp for BRSV and a 258 bp for BPIV-3 simultaneously or separately in the samples,depending on its infection status.But no specific band was amplified from other four viruses.As little as 10 pg of BVDV,1 pg of BPIV-3 and 10 pg of BRSV RNA could be detected using gel electrophoresis.Using the RT-PCR,37clinical samples were detected.The data showed that the multiple RT-PCR method was 100% coincident with the single RT-PCR,and it can be used for the detection and differential diagnosis of three viruses.%根椐GenBank中牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)、牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)和牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV-3)3种病毒基因序列,设计合成引物,建立3种病毒的三重RT-PCR方法.用这3对引物对同一样品中的BVDV、BRSV和BPIV-3核酸模板进行三重RT-PCR扩增,结果显示:可同时扩增BVDV的466 bp,BRSV的735 bp和BPIV-3的258 bp的特异性片段,而对其他4种病原的PCR扩增结果均为阴性;敏感性测定结果表明,该三重RT-PCR技术能检出10 pg的BVDV、1 pg的BHV-3和10 pg的BRSV模板.用37份临床病料对本研究多重RT-PCR技术和单项RT-PCR技术进行对比验证,结果显示:两者的总符合率为100%.结果表明:建立的多重RT-PCR检测方法,具有特异、快速、

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

    OpenAIRE

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

    2012-01-01

    The PLEX-ID/Flu assay has been recently developed to enable the detection and typing of influenza viruses based on the RT-PCR/electrospray ionization mass spectrometry technology. This novel assay was evaluated for typing performance on 201 positive influenza A or B nasopharyngeal swab specimens (NPS) detected by real-time RT-PCR during the 2010-2011 season. The PLEX-ID/Flu assay detected and characterized 91.3% and 95.3% of all influenza A and B samples, respectively. All non-typeable influe...

  20. Pelacakan Kasus Flu Burung pada Ayam dengan Reverse Transcriptase Polymerase Chain Reaction* (DETECTION OF AVIAN INFLUENZA IN CHICKENS BY REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION

    Directory of Open Access Journals (Sweden)

    Gusti Ayu Yuniati Kencana

    2013-07-01

    Full Text Available Avian Influenza (AI or Bird Flu is a fatal zoonotic disease caused by highly pathogenic avian influenza(HPAI virus of H5N1 sub-type. The disease is still endemic in Indonesia. This study was conducted toinvestigate AI cases in chickens in Bali. Virus isolation was performed in 9 day-old embryonated chickeneggs, and then followed by serologic testing by haemaglutination (HA and Haemaglutination Inhibition(HI assay using standard microtiter procedure. All of the samples were further tested with reversetrancriptasepolymerase chain reaction (RT-PCR. All work has been done in the Biomedical and MolecularBiology Laboratory, Faculty of Veterinary Medicine, Udayana University, Denpasar, during the period2009-2011. A total of ten samples were examined A total of ten chicken samples consisting of 6 fieldsamples and 4 meat samples have been confirmed to be AIV H5N1. All field cases showed clinical signsand gross pathology that were typical to the infection of avian influenza. The result indicates that AI casesare still prevalent among chickens in Bali.

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

    Science.gov (United States)

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

    2016-03-01

    HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. PMID:26709100

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

    International Nuclear Information System (INIS)

    Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent 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. 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 and the data analyzed by descriptive statistics, geNorm and NormFinder. In addition, the expression of selected reference genes in laser capture microdissected ER+ IBC cells were compared with that of whole-tissue. A group of 3 genes, TBP, RPLP0 and PUM1, were identified for both the combined group 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. The reference genes of choice when performing RT-qPCR on normal and malignant breast specimens should be either the collected group of 3 genes (TBP, RPLP0 and PUM1) employed as an average, or PUM1 as a single gene

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

    Directory of Open Access Journals (Sweden)

    Lænkholm Anne-Vibeke

    2008-01-01

    Full Text Available Abstract Background Quantitative real-time RT-PCR (RT-qPCR has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent 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 and the data analyzed by descriptive statistics, geNorm and NormFinder. In addition, the expression of selected reference genes in laser capture microdissected ER+ IBC cells were compared with that of whole-tissue. Results A group of 3 genes, TBP, RPLP0 and PUM1, were identified for both the combined group 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 and malignant breast specimens should be either the collected group of 3 genes (TBP, RPLP0 and PUM1 employed as an average, or PUM1 as a single gene.

  4. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

    Directory of Open Access Journals (Sweden)

    Porceddu Enrico

    2009-02-01

    Full Text Available Abstract Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability

  5. The chain re-action

    CERN Multimedia

    2009-01-01

    On 18 March, beam commissioning started in the first ‘link’ of the accelerator chain – LINAC 2. This marks the start of what will be the longest period of beam operations in CERN’s history, with the accelerator complex remaining operational throughout the winter to supply the LHC. The Bulletin finds out what is being done to make sure the whole chain is ready for this historic run.

  6. Detection of human enteric viruses in stream water with RT-PCR and cell culture.

    Science.gov (United States)

    Denis-Mize, K.; Fout, G.S.; Dahling, D.R.; Francy, D.S.

    2004-01-01

    A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.

  7. Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug

    Science.gov (United States)

    Bansal, Raman; Mittapelly, Priyanka; Chen, Yuting; Mamidala, Praveen; Zhao, Chaoyang; Michel, Andy

    2016-01-01

    The brown marmorated stink bug (Halyomorpha halys) has emerged as one of the most important invasive insect pests in the United States. Functional genomics in H. halys remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (RPS26, EF1A, FAU, UBE4A, ARL2, ARP8, GUS, TBP, TIF6 and RPL9) for their stability across various treatments in H. halys. Our treatments included two biotic factors (tissues and developmental stages) and two stress treatments (RNAi injection and starvation). Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). The qRT-PCR results indicated ARP8 and UBE4A exhibit the most stable expression across tissues and developmental stages, ARL2 and FAU for dsRNA treatment and TBP and UBE4A for starvation treatment. Following the dsRNA treatment, all genes except GUS showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in H. halys, we performed RNAi by administering dsRNA of target gene (catalase) through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed. PMID:27144586

  8. 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.

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

    OpenAIRE

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

    2014-01-01

    Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A ...

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2015-04-01

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

  12. Standardized RT-PCR conditions for detection and identification of eleven viruses of potato and Potato spindle tuber viroid

    Science.gov (United States)

    Standardized RT-PCR procedures were developed and validated for detection of Alfalfa mosaic virus (AMV), Impatiens necrotic spot virus (INSV), Tobacco rattle virus (TRV), Tomato spotted wilt virus (TSWV), Potato leaf roll virus (PLRV), Potato mop top virus (PMTV), Potato virus A (PVA), Potato viru...

  13. 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.

  14. 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.

  15. Detection of dengue viral RNA in Aedes aegypti (Diptera: Culicidae) exposed to sticky lures using reverse-transcriptase polymerase chain reaction.

    Science.gov (United States)

    Bangs, M J; Tan, R; Listiyaningsih, E; Kay, B H; Porter, K R

    2001-09-01

    Active surveillance for dengue (DEN) virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. However, doing so may pose logistical problems if mosquitoes must be kept alive or frozen fresh to detect DEN virus. In an attempt to simplify mosquito processing, we evaluated the usefulness of a sticky lure and a seminested reverse-transcriptase polymerase chain reaction assay (RT-PCR) for detecting DEN virus RNA under laboratory conditions using experimentally infected Aedes aegypti (L.) mosquitoes. In the first experiment, 40 male mosquitoes were inoculated with 0.13 microl of a 10(4) pfu/ml DEN-2 stock solution. After a 7-d incubation period, the mosquitoes were applied to the sticky lure and kept at room temperatures of 23-30 degrees C. Following 7, 10, 14, and 28 d application, 10 mosquitoes each were removed from the lure, pooled, and assayed for virus. DEN virus nucleic acid was clearly detectable in all pools up to 28 d after death. A second study evaluated sensitivity and specificity using one, two, and five DEN-infected mosquitoes removed after 7,10, 14, 21, and 30 d application and tested by RT-PCR. All four DEN serotypes were individually inoculated in mosquitoes and evaluated using the same procedures as experiment 1. The four serotypes were detectable in as few as one mosquito 30 d after applications to the lure with no evidence of cross-reactivity. The combination of sticky lures and RT-PCR show promise for mosquito and dengue virus surveillance and warrant further evaluation. PMID:11580045

  16. Reverse transcription and polymerase chain reaction: principles and applications in dentistry Transcrição reversa e reação em cadeia da polimerase: princípios e aplicações em odontologia

    Directory of Open Access Journals (Sweden)

    Carlos Ferreira dos Santos

    2004-03-01

    Full Text Available Various molecular biology techniques have become available in the last few years. One of the most revolutionary of these techniques regarding nucleic acid analysis is the polymerase chain reaction (PCR, which was first described in 1985. This method relies on the exponential amplification of specific DNA fragments, resulting in millions of copies that can serve as templates for different kinds of analyses. PCR can be preceded by a reverse transcription (RT reaction in order to produce cDNA from RNA (RT-PCR. RT-PCR provides the possibility to assess gene transcription in cells or tissues. PCR and RT-PCR techniques have been instrumental in dental research, and show potential to be used for diagnosis as well as for treatment and prevention of many diseases (dental caries, periodontal disease, endodontic infections and oral cancer. Compared to other traditional methodologies, PCR and RT-PCR show many advantages including high specificity, sensitivity, and speed. Since PCR and RT-PCR are relatively new techniques and are not available to most students and professionals involved with dentistry, the aim of this work is to present the details of these techniques as well as dental literature reports in which they were used.Várias técnicas de biologia molecular têm sido disponibilizadas nos últimos anos. Uma que revolucionou a análise de ácidos nucléicos foi a reação em cadeia da polimerase (PCR, descrita pela primeira vez em 1985. Esta técnica baseia-se na possibilidade de amplificação exponencial de fragmentos específicos de DNA, com a criação de milhões de cópias que servirão como matéria-prima para diferentes tipos de análises. A PCR pode ser precedida por uma reação de transcrição reversa (RT para a obtenção de cDNA a partir de RNA (RT-PCR, representando, por exemplo, uma possibilidade de análise de expressão gênica em células ou tecidos. As técnicas de PCR e RT-PCR têm sido utilizadas em pesquisas odontológicas como

  17. Factor VIII (F8) inversions in severe hemophilia A: Male germ cell origin and diagnosis with RT-PCR

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E. [Geneva Medical School (Switzerland)]|[Johns Hopkins School of Medicine, Baltimore, MD (United States); Rossiter, J.P. [Johns Hopkins School of Medicine, Baltimore, MD (United States); Young, M. [Geneva Medical School (Switzerland)] [and others

    1994-09-01

    The Factor VIII (F8) gene, which is defective in hemophilia A, is located in the most telomeric megabase of Xq. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomeric to it account for nearly half of the cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the inversion process, and that therefore the event originates predominantly in male germ cells. In all 21 informative cases in which the inversion originated in a maternal grandparent, DNA polymorphism analysis using markers within or very closely linked to F8, determined that it occurred in the male germline. In addition, all but one of 56 mothers of sporadic cases due to inversions were carriers. The data indicate that the F8 gene inversions leading to severe hemophilia A occur almost exclusively in male germ cells. The mean age of maternal grandfathers at the birth of their carrier daughters was 29.9 years (13 cases), i.e. not different from the mean paternal age in the general population, supporting the hypothesis that the inversions occur in meiosis. The inversions can be diagnosed by Southern blot analysis. For more rapid diagnosis we have used RT-PCR of RNA ectopically expressed in blood. Oligonucleotides were used to PCR amplify, after the initial RT reaction of RNA samples using random hexamers, either the normal transcript (F8 exons 21 to 24;312 bp product) or the novel abnormal transcript that is generated after the inversion. Both type 1 and 2 inversions can be recognized in affecteds and carriers by the presence of the diagnostic PcR product of 248 bp. Correct diagnoses were made in samples from 6 patients and 2 carriers with type 1 inversions, 2 patients and 2 carriers with type 2 inversions and 5 normal controls.

  18. 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

  19. 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.

  20. APPLICATION OF RT-PCR FOR ACETOBACTER SPECIES DETECTION IN RED WINE

    Directory of Open Access Journals (Sweden)

    Attila Kántor

    2014-02-01

    Full Text Available Acetic acid bacteria play a negative role in wine making because they increase the volatile acidity of wines. They can survive in the various phases of alcoholic fermentation and it is very important to control their presence and ulterior development. The main objective of the present work is to test fast, sensitive and reliable technique such as real-time PCR (rt-PCR and detecting the presence of Acetobacter aceti, Acetobacter pasteurianus, Gluconobacter oxydans, Gluconacetobacter liquefaciens and Gluconacetobacter hansenii in red wine. The aim of our study was the identification of some species of acetic acid bacteria in red wine during the fermentation process using a classical microbiological method. The changes in different groups of microorganisms were monitored in total counts of bacteria, and Acetobacter cells. Microbiological parameters were observed during the current collection and processing of wine in 2012. Samples (Modry Portugal, MP and Frankovka modra, FM were taken during the fermentation process in wine enterprises and were storage with different conditions. The total counts of bacteria ranged from 4.21 in the wine MP at 4°C of storage to 5.81 log CFU.mL-1 in the wine MP at 25°C of storage, but the total counts of bacteria ranged from 4.85 in the wine FM at 4°C of storage to 5.63 log CFU.mL-1 in the wine FM at 25°C of storage. The higher number of Acetobacter cells was found in wine MP at 25°C.

  1. 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.

  2. Avaliação da urina e de leucócitos como amostras biológicas para a detecção ante mortem do vírus da cinomose canina por RT-PCR em cães naturalmente infectados Evaluation of the urine and leucocytes as biological samples for ante mortem detection of canine distemper virus by RT-PCR assay in naturally infected dogs

    Directory of Open Access Journals (Sweden)

    F.J. Negrão

    2007-02-01

    Full Text Available Urine and leucocytes were comparatively evaluated as clinical samples for ante mortem detection of the canine distemper virus (CDV by a reverse transcription-polymerase chain reaction (RT-PCR assay. One hundred and eighty eight dogs with clinical symptoms of distemper, were distributed in three groups. The group A was constituted of 93 dogs with systemic signs of distemper; the group B by 11 dogs with neurological signs, and the group C by 84 dogs that presented simultaneously systemic and neurological signs. In 66.5% (125/188 of the dogs was amplified an amplicon with 287 base pair of the CDV nucleoprotein gene. In 60.8% (76/125 of the animals the CDV was detected simultaneously in the urine and leucocytes, and in 39.2% (49/125 of the dogs just a type of clinical sample (urine: n=37; leucocytes: n=12 was positive. These results demonstrate that the different forms of clinical distemper disease can hinder the choice of only one type of clinical sample to carry out the ante mortem etiological diagnosis of CDV infection, and false-negative results can be generated.

  3. 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

  4. Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

    DEFF Research Database (Denmark)

    Knüsel, R.; Bergmann, S. M.; Einer-Jensen, Katja;

    2007-01-01

    in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus...

  5. VARIATION OF THE EXPRESSION OF ENDOGENOUS "HOUSEKEEPING" GENES IN B[A]P TREATED MOUSE LUNGS MEASURED BY qRT-PCR

    Science.gov (United States)

    Quantitative RT-PCR is frequently used to analyze gene expression in different experimental systems. In this assay, housekeeping genes are frequently used to normalize for the variability between samples (relative quantification). We have examined the utility of using qRT-PCR and...

  6. Isolation and Differentiation of Virulent and Non-Virulent Strains of Newcastle Disease Virus by Polymerase Chain Reaction from Commercial Broiler Chicken Flocks in Shiraz-Iran

    Directory of Open Access Journals (Sweden)

    P.D. Fazel

    2012-12-01

    Full Text Available Newcastle Disease (ND is highly contagious infection of poultry that causes nervous signs and mortality in poultry. This study has been one during two years from 2010-2011 in Shiraz, Iran. The poultry industry in Fars province faced an almost heavy loss that was characterized by mild and in some flocks high mortality and respiratory distress. The aim of this study was designed to clarify the roles of the Newcastle Disease Virus (NDV by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR assay and Mean Death Time (MDT test in recent outbreak in Fars province in Iran. In virology tests, Newcastle Disease Virus (NDV was isolated from tracheal and cloacal swabs samples. Diagnosis implies the differentiation to virulent and non-virulent Newcastle disease virus. During the period of this study a total 30 commercial broiler flocks with high mortality in Fras province were visited. Samples were collected from chickens with respiratory distress. Two oligonucleotide primers, representing the sequence at the cleavage site of the F protein of both virulent and non-virulent NDV strains, respectively, were used to differentiate NDV. Using the RT-PCR was able to differentiation 6 NDV reference strains 6 of which were virulent.

  7. Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

    Directory of Open Access Journals (Sweden)

    Roberta Flávia Ribeiro Rolando

    2012-06-01

    Full Text Available This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR. A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

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

    Directory of Open Access Journals (Sweden)

    McManus Linda M

    2009-08-01

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

  9. 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

    -and-mouth disease virus (FMDV) and a set for swine vesicular disease virus (SVDV) was engineered into a full-length cDNA clone containing the RNA-2 segment of Cowpea Mosaic Virus (CPMV). After co-inoculation with a plasmid that expressed CPMV RNA-1, recombinant virus particles were rescued from cowpea plants (Vigna......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...... 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...

  10. Use of proteinase K for RT-PCR of cytokine mRNA in formalin fixed tissue

    DEFF Research Database (Denmark)

    Davies, G N; Bevan, I S; Banner, Jytte;

    1996-01-01

    formalin fixed, paraffin wax embedded material of sufficient purity for reverse transcription (RT)-PCR is described. Proteinase K treatment of formalin fixed, wax embedded tissue followed by RNA STAT-60 extraction was successful in isolating mRNA suitable for RT-PCR. Interleukin (IL)-1alpha, IL-6 and......Fresh tissue from cases of sudden infant death syndrome is becoming increasingly scarce and therefore researchers interesting in studying the aetiology of this syndrome have had to resort to archival tissue, usually in the form of paraffin wax sections. A simple method for isolating mRNA from...... tumour necrosis factor (TNF) transcripts were amplified successfully from heart, but not thyroid, kidney or liver tissue, of a patient who died following rejection of a transplanted heart, and IL-1alpha, but not IL-6 or TNF, transcripts from lung tissue of a six month old baby who died of viral pneumonia...

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

    OpenAIRE

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

    2014-01-01

    The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls acro...

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

    OpenAIRE

    Sepehrizadeh Z; Tabatabaei Yazdi M; Zarrini GH; Hashemi Bozchlou S; Khoshakhlagh P

    2008-01-01

    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 ...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    OpenAIRE

    Toth Ian K; Takle Gunnhild W; Brurberg May B

    2007-01-01

    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 o...

  15. Identification of Reference Genes across Physiological States for qRT-PCR through Microarray Meta-Analysis

    OpenAIRE

    Cheng, Wei-Chung; Chang, Cheng-Wei; Chen, Chaang-Ray; Tsai, Min-Lung; Shu, Wun-Yi; Li, Chia-Yang; Hsu, Ian C.

    2011-01-01

    Background The accuracy of quantitative real-time PCR (qRT-PCR) is highly dependent on reliable reference gene(s). Some housekeeping genes which are commonly used for normalization are widely recognized as inappropriate in many experimental conditions. This study aimed to identify reference genes for clinical studies through microarray meta-analysis of human clinical samples. Methodology/Principal Findings After uniform data preprocessing and data quality control, 4,804 Affymetrix HU-133A arr...

  16. Tailing cDNAs with terminal deoxynucleotidyl transferase in RT-PCR assays to identify ribozyme cleavage products.

    OpenAIRE

    Albuquerque-Silva, J; Houard, S.; Bollen, A.

    1998-01-01

    Polytailing a cDNA with terminal deoxynucleotidyltransferase (TdT) results in the addition of a homopolymeric sequence at its 3'-end. Here we describe the use of tailing in competitive RT-PCR assays to evaluate cleavage efficiency of ribozymes. Using a system that perfectly mimics intracellular cleavage, we were able to detect as few as 1% of cleaved moieties. Furthermore, employing primers overlapping the junction between tails and the cleaved RNA moiety in non-competitive assays, the sensit...

  17. A Rare Case of Tuberculous Pyometra in a Young Infertile Female Confirmed by mRNA-based RT-PCR

    OpenAIRE

    Megha Singhal; Renu Tanwar; Ashok Kumar; Sudha Prasad

    2012-01-01

    A 25-year-old female presented to the infertility OPD with complaints of secondary infertility and pain lower abdomen with watery discharge for the past five days. She had history of undergoing hysterosalpingography in a private hospital ten days back. The interventions included drainage of pyometra, endometrial biopsy for routine and AFB smear/ culture, confirmation of diagnosis by mRNA-based RT-PCR for detection of M. tuberculosis-specific 85B antigen gene, anti-tubercular therapy. Pyometra...

  18. 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.

  19. 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.

  20. Detection of GLV, OYDV and LYSV in potato onion (Allium cepa L.,Aggregatum group) by RT-PCR

    Institute of Scientific and Technical Information of China (English)

    XU Qijiang; CHEN Dian; TONG Youli; LI Yuhua

    2007-01-01

    Three pairs of primers were designed and synthesized from nucleotide sequences of garlic latent virus (GLV), onion yellow dwarf virus (OYDV), and leek yellow stripe virus (LYSV) by using PCR primer design software. The expected fragments about 170 bp,287 bp, and 191 bp were amplified by RT-PCR for GLV, OYDV, and LYSV, respectively in disease-infected plants of potato onion(Allium cepa L., Aggregatum group), but such fragments were not obtained from healthy-looking plants and virus-free seedlings of shoot-tips. The amplified products ofGLV, OYDV and LYSV were cloned into pGEM-T vectors, and transformed into Escherichia coli.JM109. The recombinant plasmids were obtained and sequenced. The nucleotide sequences were compared with corresponding viral nucleotide sequences reported in GenBank by performing a NCBI BLAST. The analysis showed that their homology attained 75% toplants was diluted to a series of 10-1 to 10-5 and the detection sensitivity of RT-PCR was 10-4 (about 4 ng). Thus, a method of identification and detection by RT-PCR of GLV, OYDV, and SLYV was established.

  1. 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.

  2. Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    Directory of Open Access Journals (Sweden)

    Kelly J. Henrickson

    2009-10-01

    Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The

  3. Bordetella pertussis diagnosed by polymerase chain reaction

    DEFF Research Database (Denmark)

    Birkebaek, N H; Heron, I; Skjødt, K

    1994-01-01

    The object of this work was to test the polymerase chain reaction (PCR) for demonstration of Bordetella pertussis (BP) in nasopharyngeal secretions. The method was applied to patients with recently diagnosed pertussis, as verified by BP culture. In order to test the sensitivity and specificity of...

  4. Determining Annealing Temperatures for Polymerase Chain Reaction

    Science.gov (United States)

    Porta, Angela R.; Enners, Edward

    2012-01-01

    The polymerase chain reaction (PCR) is a common technique used in high school and undergraduate science teaching. Students often do not fully comprehend the underlying principles of the technique and how optimization of the protocol affects the outcome and analysis. In this molecular biology laboratory, students learn the steps of PCR with an…

  5. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen

    OpenAIRE

    Boon-Teong Teoh; Sing-Sin Sam; Kim-Kee Tan; Jefree Johari; Juraina Abd-Jamil; Poh-Sim Hooi; Sazaly AbuBakar

    2016-01-01

    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% v...

  6. 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

    convenient and safe diagnostic procedure, since cDNA synthesis and PCR is performed sequentially without inbetween opening of the PCR-tubes, thus eliminating a substantial contamination risk. The aim of the present study was to validate a real-time OneStep RT-PCR assay for the simultaneous detection and......) 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...

  7. Comparison between specific and multiplex reverse transcription-polymerase chain reaction for detection of hepatitis A virus, poliovirus and rotavirus in experimentally seeded oysters

    Directory of Open Access Journals (Sweden)

    C Coelho

    2003-06-01

    Full Text Available Outbreaks of gastroenteritis have occurred among consumers of raw or undercooked shellfish harvested from faecally polluted waters. A multiplex reverse transcription-polymerase chain reaction (RT-PCR was applied for the simultaneous detection of hepatitis A virus (HAV, poliovirus (PV and simian rotavirus (RV-SA11 and compared with specific primers for each genome sequence. Three amplified DNA products representing HAV (192 bp, PV (394 bp and RV (278 bp were identified when positive controls were used. However, when tested on experimentally contaminated raw oysters, this method was not able to detect the three viruses simultaneously. This is probably due to the low concentration of viral RNAs present in oyster extract which were partially lost during the extracts preparation.

  8. Detection of infectious bursal disease virus in various lymphoid tissues of experimentally infected specific pathogen free chickens by different reverse transcription polymerase chain reaction assays

    DEFF Research Database (Denmark)

    Kabell, Susanne; Handberg, Kurt; Kusk, Mette;

    2005-01-01

    transcription polymerase chain reaction (RT-PCR) assays, including two recently developed strain-specific assays, were employed for detection of ribonucleic acid (RNA) from three different IBDV strains in bursa tissue samples from experimentally infected specific pathogen free chickens. The virus strains......Infectious bursal disease (IBD) is a worldwide distributed immunosuppressive viral disease in young chickens, controlled by vaccination. Emergence of several strains of IBD virus (IBDV) has created a demand for strain-specific diagnostic tools. In the present experiment, five different reverse...... included vaccine strain D78, classical strain Faragher 52/70, and the very virulent Danish strain DK01 The presence of the virus infection was confirmed by histopathologic evaluation of bursa lesions. The largest number of positive samples was obtained with a strain-specific two-step multiplex (MPX) RT...

  9. Kvantitatiiviseen qRT-PCR:ään perustuva menetelmä enterovirusten tyypittämiseksi

    OpenAIRE

    Liimatainen, Maija

    2011-01-01

    Opinnäytetyön tavoitteena oli kehittää herkkä menetelmä, jolla tuotetaan spesifinen DNA-pala suoraan virus-RNA:sta käyttäen kvantitatiivista, reaaliaikaista käänteistranskriptio-polymeraasiketjureaktiota (qRT-PCR). Optimoinnissa keskityttiin enterovirusten VP1-alueen monistamiseen. Menetelmän optimointi tehtiin vaiheittain käyttäen ensin plasmidimuotoon kloonattuja virusgenomeja (PCR-vaihe) ja sitten virus-RNA:ta, joka on eristetty tunnetuista virustyypeistä sekä ns. RNA-transkripteja, ...

  10. Viral elution and concentration method for detection of influenza A viruses in mud by real-time RT-PCR

    OpenAIRE

    Deboosere, Nathalie; Horm, Srey Viseth; Delobel, Alexandre; Gachet, Jessica; Buchy, Philippe; Vialette, Michèle

    2012-01-01

    The role of environmental reservoirs in avian influenza virus (AIV) transmission has been investigated during AIV-associated outbreaks. To date, no method has been defined for detection of AIV from mud samples. A procedure using elution and polyethylene glycol (PEG) concentration steps was designed to detect AIV by RT-PCR from 42g of raw mud, corresponding to 30g of the solid fraction of mud. RNA was recovered with MagMAX AI/ND Viral RNA Isolation kit (Ambion, Austin, TX). Three elution buffe...

  11. 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

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

    OpenAIRE

    Hollmann, B.; Wenzel, J. J.; Eis-hü binger, Am; Olschlä ger, S.; Huzly, D.; Drosten, C.; Panning, M.; Gu; nther, S.; Niller, H H; Becker, S.; Monazahian, M.; Matz, B.

    2009-01-01

    Influenza A(H1N1)v virus was first identified in April 2009. A novel real-time RT-PCR for influenza A(H1N1)v virus was set up ad hoc and validated following industry-standard criteria. The lower limit of detection of the assay was 384 copies of viral RNA per ml of viral transport medium (95% confidence interval: 273-876 RNA copies/ml). Specificity was 100% as assessed on a panel of reference samples including seasonal human influenza A virus H1N1 and H3N2, highly pathogenic avian influenza A ...

  13. Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine.

    Science.gov (United States)

    Crossland, Rachel E; Norden, Jean; Bibby, Louis A; Davis, Joanna; Dickinson, Anne M

    2016-02-01

    MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; however, extensive protocol comparisons are lacking. Furthermore, robust qRT-PCR assessment of microRNA expression within EVs is problematic, as endogenous controls (ECs) previously used in cellular samples may not be present. This study compares EV isolation and RNA extraction methods (EV precipitation reagents, RNA isolation kits and ultracentrifugation) from serum or urine samples and evaluates suitable ECs for incorporation into qRT-PCR analysis. Results were assessed by electron microscopy, nanoparticle tracking analysis and bioanalyzer concentrations. The stability of 8 ECs was compared for both serum and urine EV RNA and retrospectively validated in independent cohorts (serum n=55, urine n=50). The Life Technologies precipitation reagent gave superior serum EV recovery compared to SBI reagent, as assessed by NTA size distribution, increased RNA concentration, and lower small RNA Ct values. Similarly, the Norgen Biotek Urine Exosome RNA Isolation Kit gave improved results for urine EV isolation compared to ultracentrifugation, when determined by the same parameters. The Qiagen miRNeasy™ RNA isolation kit gave suitable serum EV RNA concentrations compared to other kits, as assessed by Bioanalyzer and small RNA qRT-PCR. Small RNAs HY3 (S.D=1.77, CoV=6.2%) and U6 (S.D=2.14, CoV=8.6%) were selected as optimal ECs for serum EV microRNA expression analysis, while HY3 (S.D=1.67, CoV=6.5%) and RNU48 (S.D=1.85, CoV=5.3%) were identified as suitable for urine studies. In conclusion, this study identifies optimal methods for isolation of serum and urine EV RNA, and suitable ECs for normalisation of qRT-PCR studies. Such reports should aid in the

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

    International Nuclear Information System (INIS)

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

  15. Simultaneous Detection of Mixed Infection of Onion yellow dwarf virus and an Allexivirus in RT-PCR for Ensuring Virus Free Onion Bulbs

    OpenAIRE

    Kumar, Sandeep; Baranwal, V. K.; Joshi, Subodh; Arya, Meenakshi; Majumder, S

    2010-01-01

    Reduced seed production in onion is associated with Onion yellow dwarf virus (OYDV), a filamentous Potyvirus. Onion is also infected with other filamentous virus particles suspected to be Allexivirus. RT-PCR was used to detect mixed infection of both the viruses in leaves and bulbs. A duplex RT-PCR was developed, which simultaneously detected the presence of these two viruses in winter (Rabi) onion bulb. In summer (Kharif) onion bulbs only Allexivirus was detected. The absence of OYDV in summ...

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

    Science.gov (United States)

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

    2013-07-26

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

  17. Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus)

    OpenAIRE

    I-Hua Chen; Lien-Siang Chou; Shih-Jen Chou; Jiann-Hsiung Wang; Jeffrey Stott; Myra Blanchard; I-Fan Jen; Wei-Cheng Yang

    2015-01-01

    Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate...

  18. 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.

  19. Circadian transitions in radiation dose-dependent augmentation of mRNA levels for DNA damage-induced genes elicited by accurate real-time RT-PCR quantification

    International Nuclear Information System (INIS)

    Molecular mechanisms of intracellular response after DNA-damage by exposure to ionizing radiation have been studied. In the case of cells isolated from living body of human and experimental animals, alteration of the responsiveness by physiological oscillation such as circadian rhythm must be considered. To examine the circadian variation in the response of p53-responsible genes p21, mdm2, bax, and puma, we established a method to quantitate their mRNA levels with high reproducibility and accuracy based on real-time reverse transcription polymerase chain reaction (RT-PCR) and compared the levels of responsiveness in mouse hemocytes after diurnal irradiation to that after nocturnal irradiation. Augmentations of p21 and mdm2 mRNA levels with growth-arrest and of puma mRNA before apoptosis were confirmed by time-course experiment in RAW264.7, and dose-dependent increases in the peak levels of all the RNA were shown. Similarly, the relative RNA levels of p21, mdm2, bax, and puma per glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also increased dose-dependently in peripheral blood and bone marrow cells isolated from whole-body-irradiated mice. Induction levels of all messages reduced by half after nighttime irradiation as compared with daytime irradiation in blood cells. In marrow cells, nighttime irradiation enhanced the p21 and mdm2 mRNA levels than daytime irradiation. No significant difference in bax or puma mRNA levels was observed between nighttime and daytime irradiation in marrow cells. This suggests that early-stage cellular responsiveness in DNA damage-induced genes is modulated between diurnal and nocturnal irradiation. (author)

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

    Science.gov (United States)

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

    2011-03-01

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

  1. A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries.

    Science.gov (United States)

    Dobhal, Shefali; Olson, Jennifer D; Arif, Mohammad; Garcia Suarez, Johnny A; Ochoa-Corona, Francisco M

    2016-06-01

    Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions. PMID:26850142

  2. 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.

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

    Science.gov (United States)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2015-10-01

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

  5. Transformational leadership: a cascading chain reaction.

    Science.gov (United States)

    Murphy, Lorraine

    2005-03-01

    Historical influences still permeate contemporary nursing practise. These are mirrored in organizational philosophies, transactional and autocratic leadership styles and disempowered staff. Whilst there is disparity amongst the theorists' definitions of leadership, there is consensus pertaining to the attributes necessary to realize effective leadership. Transformational leadership is heralded as new criterion for nurse managers, and can be achieved through training, education and professional development in key leadership competencies. To achieve a chain reaction, charismatic transformational leaders espouse intellectual stimulation and individual consideration to empower staff and enhance patient care. Nurse managers that develop and foster transformational leadership can surmount oppressive traditions and confidently navigate a complex and rapidly changing health care environment. PMID:15720482

  6. Apply Triplex RT-PCR Technology to Detect Three Potato Viruses%应用三重RT-PCR技术检测三种马铃薯病毒

    Institute of Scientific and Technical Information of China (English)

    张威; 白艳菊; 范国权; 高艳玲; 申宇; 张抒; 孟宪欣

    2015-01-01

    马铃薯X病毒(PVX)、马铃薯M病毒(PVM)和马铃薯A病毒(PVA)是导致马铃薯种薯退化的重要病毒,有时复合侵染,因此建立快速、准确检测体系尤为重要。试验从中、英文文献中查找引物,通过筛选和综合评价,每种病毒确定1对特异性引物,再通过对PCR部分Mg2+、dNTPs、TaqDNA聚合酶浓度梯度优化,最终建立了稳定的三重RT-PCR反应体系,得到长度分别为711、520、273 bp的特异性条带。应用该体系和DAS-ELISA方法同时对田间150份马铃薯毒源样品进行检测,两种方法检测结果吻合,三重RT-PCR体系的灵敏度更高、特异性更强,可以用于马铃薯田间样品检测。%Potato virus X (PVX), potato virus M (PVM) and potato virus A (PVA) are important viruses which can cause potato seed degeneration and sometimes complex infection. Therefore, it is very important to establish a fast and exact detection system. In this research, specific primer for each virus was identified by reading both Chinese and English literatures, and evaluation and selection. Then, a stable RT-PCR reaction system was established through optimized density of Mg2+, dNTPs and Taq DNA polymerase and the expected fragments of 711 bp (PVX), 520 bp (PVM) and 273 bp (PVA) were successful y amplified. This system and DAS-ELISA method were simultaneously used to detect 150 potato field samples, and the results were the same. The triplex RT-PCR system is more sensitive and specific, and could be used to detect potato field samples.

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

    Science.gov (United States)

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

    2006-01-01

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

  8. Avaliação da expressão gênica de metaloproteinases de matriz e seus inibidores em colesteatomas por amplificação de ácidos nucleicos Cholesteatoma gene expression of matrix metalloproteinases and their inhibitors by RT-PCR

    Directory of Open Access Journals (Sweden)

    Carlos Eduardo Borges Rezende

    2012-06-01

    severity. MATERIALS AND METHODS: This is a prospective study. Nineteen cholesteatoma cases at different stages were selected. RNA collected from biopsy specimens was submitted to reverse transcription polymerase chain reaction (RT-PCR for semiquantitative amplification of MMP2, MMP3, MMP9, MMP13 and TIMP1. RESULTS: Six cholesteatomas were positive for at least one of the studied genes. Four samples amplified a single gene (MMP2 or MMP13 and two samples amplified three genes (MMP2, TIMP1 and MMP3 or MMP13. No sample amplified MMP9. CONCLUSION: RT-PCR can be used to assess MMP and TIMP gene expression in cholesteatomas despite technical difficulties. Gene expression profiles could not be related to disease severity.

  9. Application of F⁺RNA Coliphages as Source Tracking Enteric Viruses on Parsley and Leek Using RT-PCR.

    Science.gov (United States)

    Shahrampour, Dina; Yavarmanesh, Masoud; Najafi, Mohammad Bagher Habibi; Mohebbi, Mohebbat

    2015-12-01

    The objective of this study was to identify sources of fecal contamination in leek and parsley, by using four different F(+)RNA coliphage genogroups (IV, I indicate animal fecal contamination and II, III indicate human fecal contamination). Three different concentrations (10(2), 10(4), 10(6) pfu/ml) of MS2 coliphage were inoculated on the surface of parsley and leek samples for detection of phage recovery efficiency among two methods of elution concentration (PEG-precipitation and Ultracentrifugation) by performing double agar layer (DAL) assay in three replications. Highest recovery of MS2 was observed in PEG method and in 10(6) inoculation concentration. Accordingly, the PEG method was used for washing and isolation of potentially contaminated phages of 30 collected samples (15 samples from the market and 15 samples from the farm). The final solutions of PEG method were tested for the enumeration of plaques by DAL assay. Total RNA was then extracted from recovered phages, and RT-PCR was performed by using four primer sets I, II, III, and IV. Incidence of F(+)RNA coliphages was observed in 12/15 (80 %) and 10/15 (66/6 %) of samples were obtained from farm and market, respectively, using both DAL and RT-PCR test methods. Different genotypes (I, II, and IV) of F(+)RNA coliphages were found in farm samples, while only genotype I was detected in market samples by using the primer sets. Due to the higher frequency of genotype I and IV, the absence of genotype III, and also the low frequency of genotype II, it is concluded that the contamination of vegetable (parsley and leek) in Neyshabour, Iran is most likely originated from animal sources. PMID:26264153

  10. Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.

    Science.gov (United States)

    Zhang, Juan; Tang, Hongju; Zhang, Yuqing; Deng, Ruyuan; Shao, Li; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

    2014-05-01

    Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis. PMID:24626784

  11. 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.

  12. Embryonic Form of Smooth Muscle Myosin Heavy Chain (SMemb/MHC-B) in Gastrointestinal Stromal Tumor and Interstitial Cells of Cajal

    OpenAIRE

    Sakurai, Shinji; Fukasawa, Tomoki; Chong, Ja-Mun; TANAKA, Akira; Fukayama, Masashi

    1999-01-01

    Myosin heavy chain (MHC) isoform expression was evaluated by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) to clarify a possible link between gastrointestinal stromal tumor (GIST) and interstitial cells of Cajal (ICCs) in the gastrointestinal (GI) tract. Using monoclonal antibodies against MHC isoforms, 18 of 27 GISTs (67%) showed immunoreactivity for non-smooth-muscle myosin or the embryonic form of MHC (SMemb), but only one tumor showed immunoreactivity f...

  13. 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 < 0.0). The sensitivity, specificity, and kappa coefficient of xTAG® RVP compared with rtRT-PCR were 84.2%, 79.6%, and 0.62%, respectively. Luminex® and rtRT-PCR detected 65 (58.0%) and 57 (50.9%) viruses in 112 NPA and 35 (34.3%) and 31 (30.4%) in 102 NPS, respectively (P < 0.01). xTAG® RVP is appropriate for detecting respiratory viruses in CAP adults. Both molecular techniques yielded better results with nasopharyngeal aspirate than swabs. J. Med. Virol. 88:1173-1179, 2016. © 2016 Wiley Periodicals, Inc. PMID:27061405

  14. Reverse Transcription Polymerase Chain Reaction-based System for Simultaneous Detection of Multiple Lily-infecting Viruses

    Directory of Open Access Journals (Sweden)

    Ji Yeon Kwon

    2013-09-01

    Full Text Available A detection system based on a multiplex reverse transcription (RT polymerase chain reaction (PCR was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV, lily mottle virus (LMoV, lily symptomless virus (LSV. Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single-or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility.

  15. Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    DEFF Research Database (Denmark)

    Balcells, Ingrid; Cirera Salicio, Susanna; Busk, Peter K.

    2011-01-01

    settings. RESULTS: We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be...... designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological...... samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in...

  16. Differentiation of canine distemper virus isolates in fur animals from various vaccine strains by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations in china

    Directory of Open Access Journals (Sweden)

    Zhao Jianjun

    2011-02-01

    Full Text Available Abstract In order to effectively identify the vaccine and field strains of Canine distemper virus (CDV, a new differential diagnostic test has been developed based on reverse transcription-polymerase chain reaction (RT-PCR and restriction fragment length polymorphism (RFLP. We selected an 829 bp fragment of the nucleoprotein (N gene of CDV. By RFLP analysis using BamHI, field isolates were distinguishable from the vaccine strains. Two fragments were obtained from the vaccine strains by RT-PCR-RFLP analysis while three were observed in the field strains. An 829 nucleotide region of the CDV N gene was analyzed in 19 CDV field strains isolated from minks, raccoon dogs and foxes in China between 2005 and 2007. The results suggest this method is precise, accurate and efficient. It was also determined that three different genotypes exist in CDV field strains in fur animal herds of the north of China, most of which belong to Asian type. Mutated field strains, JSY06-R1, JSY06-R2 and JDH07-F1 also exist in Northern China, but are most closely related to the standard virulent strain A75/17, designated in Arctic and America-2 genetype in the present study, respectively.

  17. [Investigation of human papillomavirus prevalence in women in Eskişehir, Turkey by Pap smear, hybrid capture 2 test and consensus real-time polymerase chain reaction and typing with pyrosequencing method].

    Science.gov (United States)

    Aslan, Ferhat Gürkan; Us, Tercan; Kaşifoğlu, Nilgün; Özalp, Sabit Sinan; Akgün, Yurdanur; Öge, Tufan

    2016-01-01

    Human papillomavirus (HPV) infections have a broad range of clinical spectrum from subclinical or asymptomatic infection to anogenital carcinoma. The detection of HPV-DNA and determination of the risk groups in cervical cancer (CC) screening is very important because CC is considered to be a preventable illness which is the third most common cancer type of women in the world. The aims of this study were to investigate the presence of HPV-DNA in women by two different molecular methods and to compare their results together with the results of cytology, in Eskişehir, Central Anatolia, Turkey. A total of 1081 women aged between 30-65 years, who applied to Eskişehir Early Diagnosis, Screening and Training of Cancer Center (KETEM) for screening were included in the study. Three separate cervical samples were collected simultaneously from the participants for cytologic examination and molecular studies. In the first step of the study, all cervical samples were investigated for the presence of HPV-DNA by Hybrid Capture 2 (HC2; Qiagen, Germany) method. In the second part of the study, consensus real-time polymerase chain reaction (RT-PCR) (Takara Bio Inc., Japan) was performed in 152 samples which included HC2 positive and randomly selected negative samples, and then the HPV genotypes were detected by using a commercial kit based on pyrosequencing method (Diatech Pharmacogenetics S.R.L, Italy). In the first part of the study, HC2 test was found positive in 3% (32/1081) of the women, while in 4.4% (47/1081) Pap smear was positive alone or with HC2 test. Five (0.5%) samples yielded positive results with both of the methods, and four of them were positive for high risk HPV types. Cytology results were negative in 19 out of 23 (23/1081, 2.1%) samples that were reported as high risk HPV by HC2 test. On the other hand, 42 (42/1081, 3.9%) samples that were positive by cytology yielded negative results by HC2 test. In the second part of the study, 32 (21.1%) of 152 selected

  18. Chain reaction. History of the atomic bomb

    International Nuclear Information System (INIS)

    Henri becquerel tracked down in 1896 a strange radiation, which was called radioactivity by Marie Curie. In the following centuries German scientists Max Planck, Albert Einstein and Werner Heisenberg presented fundamental contributions to understand processes in the atomic nucleus. At Goettingen, center of the international nuclear physics community, the American student J. Robert Oppenheimer admit to this physical research. In the beginning of 1939 the message of Otto Hahns' nuclear fission electrified researchers. The first step, unleashing atomic energy, was done. A half year later the Second World War begun. And suddenly being friend with and busily communicating physicians were devided into hostile power blocs as bearers of official secrets. The author tells in this exciting book the story of the first atomic bomb as a chain reaction of ideas, discoveries and visions, of friendships, jealousy and intrigues of scientists, adventurers and genius. (orig./GL)

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

    Science.gov (United States)

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

    2016-01-01

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

  20. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen.

    Science.gov (United States)

    Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Johari, Jefree; Abd-Jamil, Juraina; Hooi, Poh-Sim; AbuBakar, Sazaly

    2016-01-01

    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples. PMID:27278716

  1. P05.04A COMBINED STRATEGY FOR THE DETECTION OF BRAF FUSIONS IN PILOCYTIC ASTROCYTOMA USING RT-PCR AND FISH

    OpenAIRE

    Haynes, H.R.

    2014-01-01

    INTRODUCTION: Pilocytic astrocytomas (WHO grade I) are the most common paediatric gliomas. The FISH test for KIAA1549-BRAF fusions is most commonly used, but this is difficult to interpret. We aimed to develop a reverse-transcriptase and real-time PCR (RT-PCR) test as a first-line screen for the three most common KIAA1549-BRAF fusion variants. METHODS: A RT-PCR method for detecting KIAA1549-BRAF fusions from formalin-fixed paraffin-embedded (FFPE) brain tumour tissues (pilocytic astrocytoma) ...

  2. Reliability at the Lower Limits of HIV-1 RNA Quantification in Clinical Samples: A Comparison of RT-PCR versus bDNA Assays

    OpenAIRE

    Lubelchek, Ronald J.; Max, Blake; Sandusky, Caroline J.; Hota, Bala; Barker, David E.

    2009-01-01

    Introduction To explore whether an assay change was responsible for an increasing proportion of patients with undetectable HIV viral loads at our urban HIV clinic, we selected highly stable patients, examining their viral loads before and after changing assays. We compared the proportion with detectable viremia during RT-PCR vs. bDNA periods. Methodology/Principal Findings We selected patients with ≥1 viral loads assessed during both RT-PCR and bDNA periods. We included patients with stable C...

  3. 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...

  4. Simultaneous detection of novel H7N9 and other influenza A viruses in poultry by multiplex real-time RT-PCR

    OpenAIRE

    Xu, Xiaolong; Bao, Hongmei; Ma, Yong; Sun, Jiashan; Zhao, Yuhui; Wang, Yunhe; Shi, Jianzhong; Zeng, Xianying; Li, Yanbing; Wang, Xiurong; Chen, Hualan

    2015-01-01

    Background A novel reassortant H7N9 influenza A virus has crossed the species barrier from poultry to cause human infections in China in 2013 and 2014. Rapid detection of the novel H7N9 virus is important to detect this virus in poultry and reduce the risk of an epidemic in birds or humans. Findings In this study, a multiplex real-time RT-PCR (rRT-PCR) assay for rapid detection of H7N9 and other influenza A viruses was developed. To evaluate the assay, various influenza A viruses, other avian...

  5. A new molecular breast cancer subclass defined from a large scale real-time quantitative RT-PCR study

    International Nuclear Information System (INIS)

    Current histo-pathological prognostic factors are not very helpful in predicting the clinical outcome of breast cancer due to the disease's heterogeneity. Molecular profiling using a large panel of genes could help to classify breast tumours and to define signatures which are predictive of their clinical behaviour. To this aim, quantitative RT-PCR amplification was used to study the RNA expression levels of 47 genes in 199 primary breast tumours and 6 normal breast tissues. Genes were selected on the basis of their potential implication in hormonal sensitivity of breast tumours. Normalized RT-PCR data were analysed in an unsupervised manner by pairwise hierarchical clustering, and the statistical relevance of the defined subclasses was assessed by Chi2 analysis. The robustness of the selected subgroups was evaluated by classifying an external and independent set of tumours using these Chi2-defined molecular signatures. Hierarchical clustering of gene expression data allowed us to define a series of tumour subgroups that were either reminiscent of previously reported classifications, or represented putative new subtypes. The Chi2 analysis of these subgroups allowed us to define specific molecular signatures for some of them whose reliability was further demonstrated by using the validation data set. A new breast cancer subclass, called subgroup 7, that we defined in that way, was particularly interesting as it gathered tumours with specific bioclinical features including a low rate of recurrence during a 5 year follow-up. The analysis of the expression of 47 genes in 199 primary breast tumours allowed classifying them into a series of molecular subgroups. The subgroup 7, which has been highlighted by our study, was remarkable as it gathered tumours with specific bioclinical features including a low rate of recurrence. Although this finding should be confirmed by using a larger tumour cohort, it suggests that gene expression profiling using a minimal set of

  6. Application of One-step RT-PCR Used to Detect Bulk Milk in BVDV Eradication Programme%一步法RT—PCR检测大缸奶在BVDV清除计划中的应用

    Institute of Scientific and Technical Information of China (English)

    黄凯; 邵晓磊; 李锡智

    2011-01-01

    本文利用一步法RT—PCR对28个牛场共计50份大缸奶样进行了BVDV核酸检测。在与全群抗原检测结果对比后发现.9个已知存有PI牛的泌乳牛群,其大缸奶核酸检测均为阳性,其余41个已知无PI牛的泌乳牛群.除1个因为存在急性感染牛造成阳性结果外,其余40个均为阴性。由此可见,本方法对与泌乳牛群内是否存在PI牛可以做出准确判断,其检测灵敏度和特异性均为100%,阳性预测结果可信度为0.9(9/10),阴性预测结果可信度为0.98(40/41)。此外.针对50个大缸奶样进行的抗体检测表明,对于已感染牛场,泌乳牛群是否存在PI牛,抗体水平没有明显差异(OD1.12±0.12vsOD1.34±0.23,P〉0.05)。实验室条件下,本试验使用的RT—PCR方法对于阳性乳的最低检出限为50uL/头,因此理论上,最多可从1000份样品中检出阳性乳成分(总体积为50mL)。综上可知,本试验确立的RT—PCR方法灵敏度高、特异性强,可对泌乳牛群是否存在PI牛进行准确预测,相比大缸奶抗体检测更具实际指导意义,联合ELISA—Ag使用时还可大幅度降低泌乳牛群BVDV清除计划的检测成本,因而值得推广使用。%A single step, single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) test was developed to detect the presence of bovine viral diarrhoea virus(BVDV) in somatic cells from bulk milk samples. The test was validated by examining 50 bulk milk samples from 28 herds with result of BVDV elimination programme. Results showed that a total of 9 groups which contained one persistently infected animal were positive on bulk milk samples. Another 41 herds were negative except one herd which was positive because there was one acute infection cow. So the test was proved to be highly specific and sensitivity, which the positive predictive value(PPV) and the negative predictive value

  7. Rapid detection and characterization of Chikungunya virus by RT-PCR in febrile patients from Kerala, India.

    Science.gov (United States)

    Joseph, Anu Yamuna; Babu, Vidhu Sankar; Dev, Sona S; Gopalakrishnapai, Jayashree; Harish, M; Rajesh, M D; Anisha, S; Mohankumar, C

    2008-08-01

    There has been a resurgence and prevalence of fever with symptoms of Chikungunya (CHIK) and increased death toll in Kerala, the southern-most state of India. The objective of this study was to develop a rapid detection method to determine the presence of CHIK- virus in the serum samples collected from febrile patients in Kerala, India. Serum specimens were analyzed for CHIK viral RNA by RT-PCR using primers specific for nsP1 and E1 genes. Five out of twenty clinical samples were positive for CHIK virus. The partial sequences of the E1 and nsP1 genes of the strain, IndKL01 were highly similar to the Reunion strains and the recently isolated Indian strains. A novel substitution, A148V, was detected in the E1 gene of the isolate, IndKL02. The detection procedure used in this study was simple, sensitive and rapid (less than 4 hr). This result suggests that CHIK viruses similar to the Reunion strains, which had resulted in high morbidity and mortality rates, may have caused the recent Chikungunya outbreak in India. The effect of the variant, E1-A148V, in the virulence and the rate of transmission of the virus deserves further investigation. PMID:18814485

  8. 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. PMID:26611227

  9. RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template.

    Science.gov (United States)

    Okuda, M; Hanada, K

    2001-08-01

    RT-PCR procedures for detection of multiple species of tospovirus from plant tissues were investigated. Downstream primers were designated from the 3' untranslated sequences of the S RNA. An upstream primer was designated from the degenerated sequences of the nucleocapsid protein. Approximately 450 bp DNA fragments were detected when Tomato spotted wilt virus (TSWV)- or Impatiens necrotic spot virus (INSV)- infected tissues were examined. Approximately 350 bp DNA fragments were detected when Watermelon silver mottle virus (WSMoV)- or Melon yellow spot virus (MYSV)-infected tissues were examined. Template RNA was extracted using CF 11 cellulose powder, and nonspecific amplification became unnoticeable when double-stranded RNA was used. The amplified fragments of WSMoV were differentiated from those of MYSV by AluI or TaqI digestion. The amplified fragments of TSWV were differentiated from those of INSV by DraI or HindIII digestion. An alstroemeria plant that was infected with an unidentified tospovirus was also examined, and a 350 bp fragment that was 97% identical to Iris yellow spot virus was detected. This method, therefore, detected at least five distinct Tospovirus species. PMID:11445145

  10. Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR.

    Science.gov (United States)

    Ryu, Dong-Kyun; Ahn, Yeji; Ryu, Wang-Shick; Windisch, Marc P

    2015-11-01

    After encapsidation, where pregenomic RNA (pgRNA) is packaged into viral nucleocapsids, hepatitis B virus (HBV) uses the pgRNA as a template to replicate its DNA genome by reverse transcription. To date, there are only two encapsidation detection methods for evaluating the amount of pgRNA packaged into nucleocapsids: (i) the RNase protection assay and (ii) the native agarose gel electrophoresis assay. However, these methods are complex and laborious because they require multiple pgRNA purification steps followed by detection via an isotope-labeled probe. Moreover, both assays are unsuitable for evaluating a large number of antiviral agents in a dose-dependent manner. To overcome these limitations, we devised a novel HBV encapsidation assay in a 96-well plate format using nucleocapsid capture plates coated with an anti-HBV core (HBc) antibody, usually employed in enzyme-linked immunosorbent assays, to immobilize viral nucleocapsids. Viral pgRNA is then detected by quantitative RT-PCR (RT-qPCR). This strategy allows fast, convenient, and quantitative analysis of multiple viral RNA samples to evaluate encapsidation inhibitors. Furthermore, our protocol is potentially suitable for high-throughput screening (HTS) of compounds targeting HBV pgRNA encapsidation. PMID:26554506

  11. An 8-gene qRT-PCR-based gene expression score that has prognostic value in early breast cancer

    Directory of Open Access Journals (Sweden)

    Cejas Paloma

    2010-06-01

    Full Text Available Abstract Background Gene expression profiling may improve prognostic accuracy in patients with early breast cancer. Our objective was to demonstrate that it is possible to develop a simple molecular signature to predict distant relapse. Methods We included 153 patients with stage I-II hormonal receptor-positive breast cancer. RNA was isolated from formalin-fixed paraffin-embedded samples and qRT-PCR amplification of 83 genes was performed with gene expression assays. The genes we analyzed were those included in the 70-Gene Signature, the Recurrence Score and the Two-Gene Index. The association among gene expression, clinical variables and distant metastasis-free survival was analyzed using Cox regression models. Results An 8-gene prognostic score was defined. Distant metastasis-free survival at 5 years was 97% for patients defined as low-risk by the prognostic score versus 60% for patients defined as high-risk. The 8-gene score remained a significant factor in multivariate analysis and its performance was similar to that of two validated gene profiles: the 70-Gene Signature and the Recurrence Score. The validity of the signature was verified in independent cohorts obtained from the GEO database. Conclusions This study identifies a simple gene expression score that complements histopathological prognostic factors in breast cancer, and can be determined in paraffin-embedded samples.

  12. Development and Practical Use of RT-PCR for Seed-transmitted Prune dwarf virus in Quarantine.

    Science.gov (United States)

    Lee, Siwon; Shin, Yong-Gil

    2014-06-01

    Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea. PMID:25289000

  13. Broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex RT-PCR DNA microarray system.

    Science.gov (United States)

    Huguenin, Antoine; Moutte, Lauryane; Renois, Fanny; Leveque, Nicolas; Talmud, Deborah; Abely, Michel; Nguyen, Yohan; Carrat, Fabrice; Andreoletti, Laurent

    2012-06-01

    Newly available molecular tools allow a sensitive detection of a broad panel of viruses in respiratory tract specimens. In the present study, the application of a multiplex RT-PCR DNA microarray in diagnosis and epidemiological survey of viral infections in infants hospitalized for bronchiolitis was assessed. One hundred and thirty-eight nasopharyngeal aspirates collected from October 2007 to September 2008 were tested by direct immunofluorescence and viral culture, a combination of referenced RT-PCRs and the DNA microarray. One or more viruses were detected in 96, 126 and 126 of the specimens by direct immunofluorescence and viral culture, RT-PCRs and DNA microarray, respectively (70 vs. 91 vs. 91%, P 0.1). In conclusion, the use of this DNA microarray in clinical virology practice allows rapid and accurate identification of common and uncommon viral respiratory pathogens in infants hospitalized for bronchiolitis. It should improve the clinical management, the epidemiological survey, and the prevention of the nosocomial transmission of respiratory viruses in pediatric wards. PMID:22499022

  14. An 8-gene qRT-PCR-based gene expression score that has prognostic value in early breast cancer

    International Nuclear Information System (INIS)

    Gene expression profiling may improve prognostic accuracy in patients with early breast cancer. Our objective was to demonstrate that it is possible to develop a simple molecular signature to predict distant relapse. We included 153 patients with stage I-II hormonal receptor-positive breast cancer. RNA was isolated from formalin-fixed paraffin-embedded samples and qRT-PCR amplification of 83 genes was performed with gene expression assays. The genes we analyzed were those included in the 70-Gene Signature, the Recurrence Score and the Two-Gene Index. The association among gene expression, clinical variables and distant metastasis-free survival was analyzed using Cox regression models. An 8-gene prognostic score was defined. Distant metastasis-free survival at 5 years was 97% for patients defined as low-risk by the prognostic score versus 60% for patients defined as high-risk. The 8-gene score remained a significant factor in multivariate analysis and its performance was similar to that of two validated gene profiles: the 70-Gene Signature and the Recurrence Score. The validity of the signature was verified in independent cohorts obtained from the GEO database. This study identifies a simple gene expression score that complements histopathological prognostic factors in breast cancer, and can be determined in paraffin-embedded samples

  15. Actinobaculum suis Detection Using Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Cristina Román Amigo

    2012-01-01

    Full Text Available Actinobaculum suis is an important agent related to urinary infection in swine females. Due to its fastidious growth characteristics, the isolation of this anaerobic bacterium is difficult, thus impairing the estimation of its prevalence. The purpose of this study was to develop and test a polymerase chain reaction (PCR for the detection and identification of A. suis and then compare these results with traditional isolation methods. Bacterial isolation and PCR were performed on one hundred and ninety-two urine samples from sows and forty-five preputial swabs from boars. The results indicate that this PCR was specific for A. suis, presenting a detection limit between 1.0×101 CFU/mL and 1.0×102 CFU/mL. A. suis frequencies, as measured by PCR, were 8.9% (17/192 in sow urine samples and 82.2% (37/45 in preputial swabs. Assessed using conventional culturing techniques, none of the urine samples were positive for A. suis; however, A. suis was detected in 31.1% (14/45 of the swabs. This PCR technique was shown to be an efficient method for the detection of A. suis in urine and preputial swabs.

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

    Science.gov (United States)

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

    2016-08-01

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

  17. Development and validation of a novel Taqman-based real-time RT-PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus

    DEFF Research Database (Denmark)

    Jonstrup, Søren Peter; Kahns, Søren; Skall, Helle Frank;

    2013-01-01

    shortened and the need for maintaining expensive cell culture facilities reduced. Here we present the validation, according to OIE guidelines, of a sensitive and specific Taqman-based real-time RT-PCR. The assay detects all isolates in a panel of 79 VHSV isolates covering all known genotypes and subtypes...

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

    Science.gov (United States)

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

  19. Use of a custom RT-PCR array to analyze toxicity pathways at different life stages in Brown Norway Rat Brain following acute Toluene exposure.

    Science.gov (United States)

    To investigate the contribution of different life stages on response to toxicants, we utilized a custom designed RT-PCR array to examine the effects of acute exposure by oral gavage of the volatile organic solvent toluene (0.00, 0.65 or 1.0 glkg) in the brains of ma1e Brown Norwa...

  20. 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...

  1. High-level expression of podoplanin in benign and malignant soft tissue tumors: immunohistochemical and quantitative real-time RT-PCR analysis.

    Science.gov (United States)

    Xu, Yongjun; Ogose, Akira; Kawashima, Hiroyuki; Hotta, Tetsuo; Ariizumi, Takashi; Li, Guidong; Umezu, Hajime; Endo, Naoto

    2011-03-01

    Podoplanin is a 38 kDa mucin-type transmembrane glycoprotein that was first identified in rat glomerular epithelial cells (podocytes). It is expressed in normal lymphatic endothelium, but is absent from vascular endothelial cells. D2-40 is a commercially available mouse monoclonal antibody which binds to an epitope on human podoplanin. D2-40 immunoreactivity is therefore highly sensitive and specific for lymphatic endothelium. Recent investigations have shown widespread applications of immunohistochemical staining with D2-40 in evaluating podoplanin expression as an immunohistochemical marker for diagnosis and prognosis in various tumors. To determine whether the podoplanin (D2-40) antibody may be useful for the diagnosis of soft tissue tumors, 125 cases, including 4 kinds of benign tumors, 15 kinds of malignant tumors and 3 kinds of tumor-like lesions were immunostained using the D2-40 antibody. Total RNA was extracted from frozen tumor tissue obtained from 41 corresponding soft tissue tumor patients and 12 kinds of soft tissue tumor cell lines. Quantitative real-time PCR reactions were performed. Immunohistochemical and quantitative real-time RT-PCR analyses demonstrated the expression of the podoplanin protein and mRNA in the majority of benign and malignant soft tissue tumors and tumor-like lesions examined, with the exception of alveolar soft part sarcoma, embryonal and alveolar rhabdomyosarcoma, extraskeletal Ewing's sarcoma/peripheral primitive neuro-ectodermal tumor and lipoma, which were completely negative for podoplanin. Since it is widely and highly expressed in nearly all kinds of soft tissue tumors, especially in spindle cell sarcoma, myxoid type soft tissue tumors and soft tissue tumors of the nervous system, podoplanin is considered to have little value in the differential diagnosis of soft tissue tumors. PMID:21234520

  2. 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. PMID:27317973

  3. A meta-analysis of kidney microarray datasets: investigation of cytokine gene detection and correlation with rt-PCR and detection thresholds

    Directory of Open Access Journals (Sweden)

    Stegall Mark D

    2007-03-01

    Full Text Available Abstract Background Microarrays provide a means to simultaneously examine the gene expression of the entire transcriptome in a single sample. Many studies have highlighted the need for novel software and statistical approaches to assess the measured gene expression. Less attention has been directed toward whether genes considered undetectable by microarray can be detected by other strategies or whether these genes can provide accurate gene expression determinations. In the kidney this is a concern for genes such as cytokines which dramatically influence the immune response but are often considered low abundance genes produced by a small number of cells. Results Using both publicly available and our own microarray datasets we analyzed the detection p-value and detection call values for 81 human kidney samples run on the U133A or U133Plus2.0 Affymetrix microarrays (Affymetrix, Santa Clara, CA. For the cytokine genes, the frequency of detection in each sample group (normal, transplant and renal cell carcinoma was examined and revealed that a majority of cytokine related genes are not detectable in human kidney by microarray. Using a subset of 29 Mayo transplant samples, a group of seven transplant-related cytokines and eight non-cytokine genes were evaluated by real-time PCR (rt-PCR. For these 15 genes we compared the impact of decreasing microarray detection frequency with the changes in gene expression observed by both microarray and rt-PCR. We found that as microarray detection frequency decreased the correlation between microarray and rt-PCR data also decreased. Conclusion We conclude that, when analyzing microarray data from human kidney samples, genes generally expressed at low abundance (i.e. cytokines should be evaluated with more sensitive approaches such as rt-PCR. In addition, our data suggest that the use of detection frequency cutoffs for inclusion or exclusion of microarray data may be appropriate when comparing microarray and rt-PCR

  4. 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.

  5. Molecular detection of host cytokine expression in Helicobacter pylori infected patients via semi-quantitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Hosseini M

    2010-01-01

    Full Text Available Background: Helicobacter pylori (Hp is a bacterium recognised as a main causative agent for the development of chronic active gastritis, peptic ulcer disease, gastric adenocarcinoma and primary gastric lymphoma. Objective: Determination of the levels of IFN-γ (pro-inflammatory and IL-4 (anti inflammatory cytokine expression as indicators of Th1 and Th2 immune responses in gastric cancer (GC and non gastric cancer (Non GC dyspeptic patients by gene specific RT-PCR. Materials and Methods: Biopsy specimens were collected from three groups of gastric cancer (GC=18, non ulcer dyspepsia (NUD = 38 and peptic ulcer patients (PUD=20. Total RNA was extracted and complementary DNA was synthesised. PCR amplification was performed for HPRT, IFN-γ and IL-4 cytokines and the intensity of each band was measured by densitometry and normalized against HPRT expression as a house keeping gene. Results: Comparison of the results from different groups of patients indicated that IFN-γ gene expression was similar in nonGC dyspeptic patients (NUD and PUD groups; 3.38 ± 0.57,3.43 ± 0.41, respectively whereas, in GC patients, it was significantly higher than others (5.52 ± 0.59; P < 0.0001. On the other hand, IL-4 gene expression showed no significant difference between NUD and GC patients (2.81 ± 0.43,2.3 ± 0.12 respectively, whereas the expression rate of this cytokine was significantly higher in PUD patients (3.7 ± 0.1; P 0.05. Our data indicate an association between Th1 and Th2 immune responses and the development of gastric cancer and peptic ulcer disease respectively.

  6. Quantification of mRNA in Salmonella sp. seeded soil and chicken manure using magnetic capture hybridization RT-PCR.

    Science.gov (United States)

    Jacobsen, Carsten Suhr; Holben, William E

    2007-05-01

    Direct quantification of mRNA from Salmonella sp. seeded for 1 h to soil and chicken manure was accomplished using magnetic capture hybridization as a purification technique. This detection strategy targeted the invA gene present in Salmonella sp. After cell lysis, phenol/chloroform purification and isopropanol precipitation, the RNA extract was combined with the hybridization probe conjugated to paramagnetic beads. After hybridization, the captured nucleic acids were released by denaturation and purified of contaminating DNA using DNase. The resulting RNA was of high purity and there was no need for dilution of the samples prior to RT-PCR. The developed procedure was reproducibly used to quantify Salmonella sp. in high organic agricultural soil. The detection limit for mRNA using ordinary quantitative PCR (employing SYBRgreen-based detection) was 5 x 10(4)Salmonella sp. cells per gram of soil. Chicken manure amended into soil (1:4 w/w) did not reduce the ability to quantify Salmonella sp. mRNA in soil. Pasteurization (65 degrees C, 30 min) of chicken manure containing Salmonella sp. dramatically reduced the detection of invA mRNA (requiring 42 qPCR cycles for detection versus 26 cycles in unpasteurized manure), presumably due to degradation of the invA mRNA in Salmonella sp. cells killed by pasteurization. By contrast, DNA-based qPCR still detected Salmonella sp. in the pasteurized manure. Thus, in this case using samples seeded with fresh Salmonella sp. the mRNA-based detection appears to be superior to minimizing false-positive detection which was prevalent with DNA-based qPCR. PMID:17383760

  7. Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes

    Directory of Open Access Journals (Sweden)

    Silva A.M.

    1999-01-01

    Full Text Available The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-g. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31% were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38% showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.

  8. 牛肠道病毒 RT -PCR 检测方法的建立及初步应用%Establishment and Application of RT-PCR for Detection of Bovine Enterovirus

    Institute of Scientific and Technical Information of China (English)

    侯佩莉; 程洪兵; 刘晓; 王洪梅; 何洪彬

    2014-01-01

    A reverse transcription PCR (RT-PCR) method for detection of bovine enterovirus (BEV) was established using a pair of specific primers designed based on the 3 D gene of BEV in GenBank .This method could specifically amplify a 732-bp fragment from BEV strain , but not from bovine viral diarrhea vi-rus (BVDV), infectious bovine rhinotracheitis virus (IBRV), bovine coronavirus (BCoV) and bovine rota-virus virus (BRV).The detection limit of this method was 10 -1 TCID50 of BEV.In a detection of 84 samples suspected with bovine disease , 21 samples were positive with the positive rate as 25%.%参照GenBank中登录的牛肠道病毒( BEV )全基因组序列,针对其3 D基因设计合成了1对特异性引物,提取病毒RNA,逆转录为cDNA,进行PCR扩增,经条件优化,建立了BEV的RT-PCR检测方法,并对该方法的特异性、敏感性进行验证。结果显示,该方法从分离的牛肠道病毒中扩增出了732 bp的特异性目的片段;且对牛传染性鼻气管炎病毒( IBRV)、牛病毒性腹泻病毒( BVDV)、牛轮状病毒( BRV)、牛冠状病毒( BCoV)等相关病毒均无交叉反应;其检出敏感度达10-1 TCID50。应用该方法对山东地区84份临床疑似发病牛样品进行检测,21份为阳性,阳性检出率为25%。

  9. RT-PCR assay for the detection of infective (L3 larvae of lymphatic filarial parasite, Wuchereria bancrofti, in vector mosquito Culex quinquefasciatus

    Directory of Open Access Journals (Sweden)

    K.P. Patra

    2008-08-01

    Full Text Available Background & objectives: Periodic monitoring of vector population for infection and infectivity rates is central to the evaluation of the filariasis elimination strategies in endemic areas to monitor the success of MDA and also to establish endpoints for intervention. The main objective of this study was to develop a RT-PCR assay, based on L3 stage-specific primers to detect the presence of infective stage larvae of filarial parasite, Wuchereria bancrofti in the vector Culex quinquefasciatus. Material & Methods: Subtracted probe development technique was employed for the identification of infective stage (L3 specific genes. The subtracted cDNA was labeled by non-radioisotopic method and used for screening cDNA library of L3 stage larvae of W. bancrofti constructed in UniZap XR. Recombinants were probed and identified from the library. The inserts of the recombinant clones were purified and sequenced. Primers were designed based on the sequence information of three recombinant clones for detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay. Preliminary laboratory evaluation was carried out to assess the sensitivity and specificity of WbL31 RT-PCR assay.Results: cDNA library of L3 stage of W. bancrofti constructed in UniZap XR vector, constituted 5×105 phages with 80–90% recombinant phages and the size of inserts varied from 0.1 to 1.0 kb. When subtracted cDNA was random prime labeled and used for screening cDNA library of L3 stage of W. bancrofti constructed in UniZap XR, 18 clones were identified from the library. Three genes were found up-regulated in the L3 stage, out of which WbL31 (cuticular collagen was found to be useful in detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay with high specificity and sensitivity (98–100% . Conclusion: Present paper marks first report on the development of an infective stage-specific RT-PCR assay (WbL31 RT-PCR assay to detect L3 stage W. bancrofti in the vector. This assay

  10. Fluorescent in situ RT-PCR to visualise the expression of a phosphate transporter gene from an ectomycorrhizal fungus.

    Science.gov (United States)

    van Aarle, Ingrid M; Viennois, Gaëlle; Amenc, Laurie K; Tatry, Marie-Violaine; Luu, Doan T; Plassard, Claude

    2007-09-01

    Expression of a mycorrhizal fungal-specific phosphate (P) transporter gene (HcPT1) was studied in mycelium of the ectomycorrhizal fungus Hebeloma cylindrosporum, by in situ reverse transcriptase polymerase chain reaction using amplification of complementary DNA sequences. The expression of HcPT1 was visualised under two different P treatments. Mycelium was transferred to liquid medium with or without P and incubated for 5 days. Under P starvation, mycelium growth and vitality was reduced and the expression of HcPT1 up regulated. Enzyme-labelled fluorescent substrate was used to detect gene expression in situ with epi-fluorescence microscopy and to visualise it at the level of the individual hyphae both in starved and non-starved hyphae. Up-regulation of HcPT1 was observed as a more intense fluorescent signal and from the larger proportion of hyphae that showed expression. PMID:17520293

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

    Science.gov (United States)

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

  12. 联合应用细胞遗传学、巢式RT-PCR和FISH技术监测慢性髓细胞白血病干扰素治疗中肿瘤负荷%Detection of the tumor load in chronic myeloid leukemia during treatment with interferon(IFN)by conventional cytogenetics,nested-RT-PCR and FISH

    Institute of Scientific and Technical Information of China (English)

    李国霞; 王慧萍; 乔振华; 王宏伟; 戴欣

    2008-01-01

    Objective To explore the sensitivity and specificity of conventional eytogeneties(CC),nested-reverse transcriptase polymerase chain reaction(nested-RT-PCR) and dual-color and dual-fusion fluorescence in situ hybridization(D-FISH) technique in monitoring the tumor load of chronic myeloid leukemia (CML) during treatment.Methods CC,nested-RT-PCR and interphase D-FISH were simultaneously carried out to detect the tumor load of 5 CML patients during treatment with interferon-Mpha(IFN-α).Results 24 specimens from 5 CML patients before and after IFN-α therapy were investigated and the results showed that 23 specimens were Ph+ with different positive ratios by CC.All specimens were bcl-abl mRNA (+) by RTPCR.The Ph-ber-abl+ specimen from case 2 after 75 months of post-treatment showed 4.5%bcr-abl+ cells by FISH.2 specimens from case 1 at 22 mortths,26 mortths of post-treatment.2 specimens from cage 5 after 12 months,16 menths of post-treatment and 2 specimens from case 4 after 6 menths,10 menths post-treatment with same Ph+ ratio respectively were investigated by FISH and showed 47.5% and 39.5%,74.0%and 60.5%,99.0% and 99.5% bcr-abl+ cells,respectively.Conclusion CC can be used as a basic tool to monitor the change of tumor load in CML during treatment. When CC can't evaluate precisely dynamic changes of tumor load and when tumor load in patient with treatment were too low to detect Ph bv CC while bcr-abl mRNA was still positive by RT-PCR,FISH Call be used to detect precisely tumor load and monitor dynamic change of the disease.More sensitive RT-PCR was used to monitor tumor load when it was negative to bcr-abl by FISH during treatment.%目的 探讨常规细胞遗传学(CC)、巢式反转录-聚合酶链反应(RT-PCR)及双色双融合荧光原佗杂交(D-FISH)三种技术监测慢性髓细胞白血病(CML)患者干扰素(INF)治疗过程中肿瘤负荷的灵敏度和特异性.方法 联合应用CC、巢式RT-PCR和D-FISH三种技术对5例INF治疗中CML患者的肿瘤

  13. 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

    OpenAIRE

    Lopez, J.C.; R Mcfarlane; Ulloa, J.

    2006-01-01

    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 compara...

  14. Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions.

    Directory of Open Access Journals (Sweden)

    Dung Tien Le

    Full Text Available Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue- and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used.

  15. Development of sensitive, high-throughput one-tube RT-PCR-enzyme hybridisation assay to detect selected bacterial fish pathogens.

    Science.gov (United States)

    Wilson, T; Carson, J

    2003-03-31

    Bacterial monitoring and surveillance is critical for the early detection of pathogens to avoid the spread of disease. To facilitate this, an efficient, high-performance and high-throughput method to detect the presence of femotgram amounts of ribosomal RNA from 4 bacterial fish pathogens: Aeromonas salmonicida; Tenacibaculum maritimum (formerly Flexibacter maritimus); Lactococcus garvieae; and Yersinia ruckeri was developed. The system uses NucleoLink strips for liquid- and solid-phase PCR in 1 tube, to perform RT-PCR-enzyme hybridisation assays (RT-PCR-EHA) detecting 4 fg or less of rRNA from pure cultures and between 1 and 9 CFU per 200 microl sample volume from selective-enrichment culture media. The liquid-phase amplicons were visualised by gel electrophoresis and the solid-phase amplicons detected using internal probes and visualised using colorimetric detection and p-nitrophenylphosphate. PMID:12747638

  16. 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...... the 3D gene of FMDV. The assay was validated for the efficacy to detect all known FMDV serotypes. The test method was linear over a range of at least 7 orders of magnitude and the detection limit was below the equivalent of 10 genomic copies. Analysing recent African probang samples the method was able...... to detect FMDV in materials from both cattle and buffalo. When compared to traditional virus cultivation the virus detection sensitivity was similar but the RT-PCR method can provide a laboratory result much faster than virus cultivation. The real-time PCR method confirms the identity of the amplicon...

  17. Semi-Nested Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Successful Quantitation of Cytokeratin mRNA Expression Levels for the Subtyping of Non-Small-Cell Lung Carcinoma Using Paraffin-Embedded and Microdissected Lung Biopsy Specimens

    International Nuclear Information System (INIS)

    In patients with inoperable advanced non-small cell lung carcinomas (NSCLCs), histological subtyping using small-mount biopsy specimens was often required to decide the indications for drug treatment. The aim of this study was to assess the utility of highly sensitive mRNA quantitation for the subtyping of advanced NSCLC using small formalin fixing and paraffin embedding (FFPE) biopsy samples. Cytokeratin (CK) 6, CK7, CK14, CK18, and thyroid transcription factor (TTF)-1 mRNA expression levels were measured using semi-nested real-time quantitative (snq) reverse-transcribed polymerase chain reaction (RT-PCR) in microdissected tumor cells collected from 52 lung biopsies. Our results using the present snqRT-PCR method showed an improvement in mRNA quantitation from small FFPE samples, and the mRNA expression level using snqRT-PCR was correlated with the immunohistochemical protein expression level. CK7, CK18, and TTF-1 mRNA were expressed at significantly higher levels (P<0.05) in adenocarcinoma (AD) than in squamous cell carcinoma (SQ), while CK6 and CK14 mRNA expression was significantly higher (P<0.05) in SQ than in AD. Each histology-specific CK, particularly CK18 in AD and CK6 in SQ, were shown to be correlated with a poor prognosis (P=0.02, 0.02, respectively). Our results demonstrated that a quantitative CK subtype mRNA analysis from lung biopsy samples can be useful for predicting the histology subtype and prognosis of advanced NSCLC

  18. Tick-borne encephalitis virus-specific RT-PCR - a rapid test for detection of the pathogen without viral RNA purification

    Czech Academy of Sciences Publication Activity Database

    Rudenko, Natalia; Golovchenko, Maryna; Cihlářová, Věra; Grubhoffer, Libor

    2004-01-01

    Roč. 48, č. 3 (2004), s. 167-171. ISSN 0001-723X R&D Projects: GA ČR GA524/03/1326 Institutional research plan: CEZ:AV0Z6022909 Keywords : Ixodes ricinus * tick-borne encephalitis virus * RT-PCR Subject RIV: GJ - Animal Vermins ; Disease s, Veterinary Medicine Impact factor: 0.605, year: 2004

  19. Sensitive detection of Tomato ringspot virus by real-time TaqMan RT-PCR targeting the highly conserved 3'-UTR region.

    Science.gov (United States)

    Tang, Joe; Khan, Subuhi; Delmiglio, Catia; Ward, Lisa I

    2014-06-01

    A real-time TaqMan RT-PCR assay was developed for the rapid and sensitive detection of Tomato ringspot virus (ToRSV), an important plant virus which infects a wide range of fruit and ornamental crops. Primers and a probe were designed based on the highly conserved 3'-untranslated region (UTR) sequences of ToRSV, to amplify a 182bp fragment of this region of RNA-1 and RNA-2. The assay was demonstrated to reliably amplify all ToRSV isolates tested. The detection limit was estimated to be about 12 copies of the ToRSV target region. No amplification was observed from the RNA of other nepoviruses or healthy host species. A comparison with a published conventional RT-PCR and a SYBR-based qRT-PCR indicated that both of the published assays lacked reliability and sensitivity, as neither were able to amplify all ToRSV isolates tested, and both were approximately 1000 times less sensitive than the novel TaqMan real-time assay. This TaqMan real-time assay was tested using four different reagent kits and was shown to be robust and stable, with no significant differences in sensitivity between kits. It is expected that the implementation of this TaqMan real-time RT-PCR assay will facilitate efficient phytosanitary certification of nursery stock requiring testing for ToRSV by regulatory agencies, and will also have wider uses for the general detection of ToRSV in a range of hosts. PMID:24566000

  20. RT-PCR assay for the detection of infective (L3) larvae of lymphatic filarial parasite, Wuchereria bancrofti, in vector mosquito Culex quinquefasciatus

    OpenAIRE

    K.P. Patra; Hoti, S.L.; V. Vasuki

    2008-01-01

    Background & objectives: Periodic monitoring of vector population for infection and infectivity rates is central to the evaluation of the filariasis elimination strategies in endemic areas to monitor the success of MDA and also to establish endpoints for intervention. The main objective of this study was to develop a RT-PCR assay, based on L3 stage-specific primers to detect the presence of infective stage larvae of filarial parasite, Wuchereria bancrofti in the vector Culex quinquefasciatus....

  1. Characterization of Human Coronavirus Etiology in Chinese Adults with Acute Upper Respiratory Tract Infection by Real-Time RT-PCR Assays

    OpenAIRE

    Lu, Roujian; Yu, Xiaoyan; Wang, Wen; Duan, Xijie; Zhang, Linglin; Zhou, Weimin; Xu, Jin; Xu, Lingjie; Hu, Qin; Lu, Jianxin; Ruan, Li; Wang, Zhong; Tan, Wenjie

    2012-01-01

    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 col...

  2. Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86

    OpenAIRE

    Jongejan Frans; Postigo Milagros; Balk Jesper A; Nijhof Ard M

    2009-01-01

    Abstract Background For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus ...

  3. RT-PCR versus immunohistochemistry for correlation and quantification of ERCC1, BRCA1, TUBB3 and RRM1 in NSCLC

    DEFF Research Database (Denmark)

    Garcia-Foncillas, J; Huarriz, M; Santoni-Rugiu, E; Sorensen, J B; Vilmar, Adam Christian

    2012-01-01

    Customized chemotherapy is increasingly used in the management of patients with advanced non-small cell lung cancer (NSCLC). However, the most reliable methodology to determine biomarker status is neither fully elucidated nor agreed upon. Accordingly, we evaluated the predictive efficiency of qRT......RT-PCR and immunohistochemical analysis (IHC) on excision cross complementation group 1 (ERCC1), breast cancer susceptibility gene 1 (BRCA1), ribonucleotide reductase subunit M1 (RRM1) and class III ß-tubulin (TUBB3)....

  4. Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors

    OpenAIRE

    TAN, Suat Cheng; Carolyn A Carr; Yeoh, Kar Kheng; Schofield, Christopher J.; Davies, Kay E.; Clarke, Kieran

    2011-01-01

    Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Her...

  5. Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

    OpenAIRE

    Ray, Debashree L; Joshua C Johnson

    2014-01-01

    Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the r...

  6. Localisation of Abundant and Organ-Specific Genes Expressed in Rosa hybrida Leaves and Flower Buds by Direct In Situ RT-PCR

    Directory of Open Access Journals (Sweden)

    Agata Jedrzejuk

    2012-01-01

    Full Text Available In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by using in situ RT-PCR in Rosa hybrida flower buds and leaves. As target genes, we used the abundant β-actin and RhFUL gene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.

  7. Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus).

    Science.gov (United States)

    Chen, I-Hua; Chou, Lien-Siang; Chou, Shih-Jen; Wang, Jiann-Hsiung; Stott, Jeffrey; Blanchard, Myra; Jen, I-Fan; Yang, Wei-Cheng

    2015-01-01

    Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. PMID:26486099

  8. Detecção do gene da nucleoproteína do vírus da cinomose canina por RT-PCR em urina de cães com sinais clínicos de cinomose Detection of canine distemper virus nucleoprotein gene by RT-PCR in urine of dogs with distemper clinical signs

    Directory of Open Access Journals (Sweden)

    C.M.S. Gebara

    2004-08-01

    Full Text Available A presença do vírus da cinomose canina (CDV foi avaliada pela reação em cadeia da polimerase, precedida de transcrição reversa (RT-PCR, em 87 amostras de urina de cães que apresentavam sinais clínicos sugestivos de cinomose. Os animais foram distribuídos em três grupos. No grupo A foram incluídos 41 cães com alterações sistêmicas; no grupo B, 37 cães com alterações neurológicas; e no grupo C, nove cães com alterações sistêmicas e neurológicas simultâneas. O grupo D (controle foi composto por 20 cães assintomáticos. Os resultados da RT-PCR foram correlacionados com a forma clínica da infecção e com as alterações hematológicas encontradas. Foi possível a amplificação parcial do gene da nucleoproteína do CDV em 41 (47,1% das 87 amostras de urina provenientes de cães com sinais clínicos sugestivos de cinomose. Todas as amostras obtidas de animais assintomáticos foram negativas na RT-PCR. Amostras positivas foram encontradas nos três grupos de animais com sinais clínicos na proporção de 51,2% (24/41, 29% (11/37 e 100% (9/9 para os grupos A, B e C, respectivamente. A leucocitose foi a alteração hematológica mais freqüente nos três grupos de cães com sinais clínicos porém, não foi possível estabelecer correlação entre o resultado da RT-PCR e as alterações hematológicas. Os resultados demonstraram que, independente da forma de apresentação clínica, a técnica da RT-PCR realizada em urina pode ser utilizada no diagnóstico ante mortem da infecção pelo CDV.The urine of 87 dogs with clinical signs suggestive of canine distemper was analyzed by RT-PCR for detection of canine distemper virus (CDV nucleoprotein gene. The samples were allotted to the following groups: group A- with 41 dogs with systemic symptoms, group B- with 37 dogs with neurological signs, and group C- with 9 dogs with simultaneous systemic and neurological clinical signs. Group D (control included 20 assymptomatic dogs. A chi2

  9. Identification of HIV-1 Genotypic Resistance in Patients on First-line Antiretroviral Therapy Using Polymerase Chain Reaction and Sequencing

    Institute of Scientific and Technical Information of China (English)

    Jiang Xiao; Gui-ju Gao; Hong-xin Zhao; Yan-mei Li; Ying-xiu Huang; Wen Zhang; Wen-jing Su; Wei Zhang; Ning Han; Di Yang; Xin Li

    2013-01-01

    Objective The aim of the study was to evaluate the characteristics of HIV drug-genotypic resistance among patients taking ifrst-line ARV regimens using polymerase chain reaction and sequencing, and guide to design optimal ARV regimens for these patients. Methods HIV reverse transcriptase-encoded gene was ampliifed with RT-PCR and ampliifed PCR products were aligned and comparatively analyzed with HIV resistance database to ifnd drug-resistance mutations. Results Twenty-eight PCR products were amplified and sequenced successfully in 30 serum samples of recruited HIV-infected patients with virologic failure. The resistance rate was 96%, mutations in NRT region were found in 26 patients (93%), while mutations in NNRT region were found in 27 patients (96%). M184V was the most common mutation (86%), K65R was selected in 14%of recruited individuals and TAMs occurred in 50%of patients, which resulted in resistance to NRTIs. Y181C and V179D were the most common mutations in NNRTIs and prevalence was 43%(12/28) and 36%(10/28), respectively, which resulted in cross-resistance to NNRTIs due to low-genetic barrier. Conclusions Virologic failure may occur in long-term administration of ifrst-line ARV regimens, and drug-resistance mutations can be found in these patients, which resulted in resistance to ifrst-line ARV regimens. We emphasized that HIV viral load assay and resistance assay were important tools to guide healthcare workers to design an optimal second-line ARV regimens for HAART-experienced individuals with virologic failure.

  10. Detection of pathogenic Yersinia enterocolitica in slaughtered pigs by cultural methods and real-time polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Rina Mazzette

    2015-05-01

    Full Text Available Healthy pigs carrying pathogenic to human Yersinia enterocolitica strains are the main source of entry into slaughterhouse, where cross-contamination of carcasses can happen. The aim of this work was to determine Y. enterocolitica prevalence in slaughtered pigs, investigating the presence of carriers in relation to carcass contamination. A total of 132 pig samples (tonsils, mesenteric lymph nodes, colon content, carcass surface were collected from 4 Sardinian slaughterhouses. All the samples were examined by the ISO 10273:2003 method, and the prevalence was also determined by direct plating on CIN Agar. Moreover, to detect the ail positive Y. enterocolitica strains in enrichment broths and isolates a real-time polymerase chain reaction (PCR was applied. Y. enterocolitica prevalence was 19% with direct plating and 12% with enrichment methods. Carcass surfaces and tonsils prevalence was 5.30% by direct plating, and 5.3% and 2.2%, respectively, by enrichment method. Tonsil samples showed an average contamination level of 3.2×103 CFU/g, while the mean value on carcass was 8.7×102 CFU/g. An overall prevalence of 9.8% of ail positive Y. enterocolitica broths was detected by RT-PCR, that found a higher prevalence in tonsils (7.5% with respect to cultural methods, confirming the greater sensitivity of this technique when applied for tonsils and faeces samples. The results show a relatively low pathogenic Y. enterocolitica prevalence in pigs slaughtered in Sardinia. Good hygiene measures should be applied at slaughterhouse in order to prevent the entry of carriers and control carcass contamination.

  11. Development and application of reverse transcriptase nested polymerase chain reaction test for the detection of exogenous avian leukosis virus.

    Science.gov (United States)

    García, Maricarmen; El-Attrache, John; Riblet, Sylva M; Lunge, Vagner R; Fonseca, André S K; Villegas, Pedro; Ikuta, Nilo

    2003-01-01

    A polymerase chain reaction (PCR) assay that utilizes nested primers to amplify a fragment of the long terminal repeat of exogenous avian leukosis virus (ALV) was developed and evaluated for detection of ALV subgroup J directly from clinical samples. Compilation of sequence data from different endogenous and exogenous ALVs allowed the selection of a conserved set of nested primers specific for the amplification of exogenous ALV subgroups A, B, C, D, and J and excluded amplification of endogenous viruses or endogenous viral sequences within the chicken genome. The nested primers were successfully used in both PCR and reverse transcriptase (RT)-PCR assays to detect genetically diverse ALV-J field isolates. Detection limits of ALV-J isolate ADOL-Hc1 DNA by nested PCR and RNA by RT-nested PCR were superior to detection of group-specific antigen by enzyme-linked immunosorbent assay (ELISA) in cell culture. Detection of ALV-J in cloacal swabs by RT-nested PCR was compared with direct detection by antigen-capture (ac)-ELISA; RT-nested PCR detected fewer positive samples than ac-ELISA, suggesting that RT-nested PCR excluded detection of endogenous virus in clinical samples. Detection of ALV-J in plasma samples by RT-nested PCR was compared with virus isolation in C/E chicken embryo fibroblasts; the level of agreement between both assays as applied to plasma samples ranged from low to moderate. The main disagreement between both assays was observed for a group of plasma samples found positive by RT-nested PCR and negative by virus isolation, suggesting that RT-nested PCR detected ALV-J genome in plasma samples of transiently or intermittently infected birds. ALV-J transient and intermittent infection profiles are characterized by inconsistent virus isolation responses throughout the life of a naturally infected flock. PMID:12713157

  12. Detection of Francisella tularensis in blood by polymerase chain reaction.

    OpenAIRE

    Long, G W; Oprandy, J J; Narayanan, R. B.; Fortier, A H; Porter, K R; Nacy, C.A.

    1993-01-01

    We developed a polymerase chain reaction-based assay for Francisella tularensis which we evaluated by using spiked blood samples and experimentally infected mice. The assay detected both type A and type B F. tularensis at levels equivalent to one CFU/microliter of spiked blood. Results from polymerase chain reaction-based assay of limiting dilutions of blood from mice infected with the live vaccine strain agreed closely with results from blood culture.

  13. CONVERGENCE OF MARKOV CHAIN APPROXIMATIONS TO STOCHASTIC REACTION DIFFUSION EQUATIONS

    OpenAIRE

    Kouritzin, Michael A.; Hongwei Long

    2001-01-01

    In the context of simulating the transport of a chemical or bacterial contaminant through a moving sheet of water, we extend a well-established method of approximating reaction-diffusion equations with Markov chains by allowing convection, certain Poisson measure driving sources and a larger class of reaction functions. Our alterations also feature dramatically slower Markov chain state change rates often yielding a ten to one-hundred-fold simulation speed increase over the previous version o...

  14. The Analgesic Effects of Apitoxin and its Mechanism via JOR and Measuring Expression of mRNA in Phospholipase and TPH using RT-PCR

    Directory of Open Access Journals (Sweden)

    Cho Kwang Ho

    2000-07-01

    Full Text Available The purpose of this study is to prove the analgesic effects of apitoxin and its mechanism via jaw-opening reflex(JOR and measuring expression of mRNA in Phospholipase and Tryptophan hydroxylase(TPH using RT-PCR. The experiments were carried out on Sprague-Dawley rats(300-400g and mastocytoma (P-185 HTR for JOR and RT-PCR, respectively. Rats anesthetized with thiopental sodium (80mg/kg were used in the Tooth Pulp stimulation induced JOR. The amplitude of a digastric electromyogram (dEMG was recorded during the stimulation at an intensity of 1.5 times the threshold for JOR. Apitoxin used in this experiment was diluted with normal saline by 1:1000. Apitoxin was injected intravenously into the test group while normal saline to the control group. However, it was injected directly into the cell of mastocytoma. We referred to base sequence registered in Genbank in designing primers for RT-PCR. The results were as follows; (1Compared with control group, analgesic effect started to show right after Sprague-Dawely rats were treated with apitoxin(71.50±8.08 and lasted for 50 minutes. (2As a result of the experiment of RT-PCR, we witnessed significant changes in the degree of expression of phospholipase or rate-limiting enzyme in biosynthesis of prostaglandins with 10μg/㎖ apitoxin.(31.74±18.98%, P<0.05 (3As a result of the experiment of RT-PCR, we witnessed significant changes in the degree of expression of TPH or rate-limiting enzyme in biosynthesis of serotonin with 10μg/㎖ apitoxin.(131.37±16.87%, P<0.05. These results suggest that 10μg/㎖ apitoxin have the most analgesic effects. This study showed that apitoxin has analgesic effects and held good for 50 minutes. The injection of apitoxin has brought out changes in the degree of expression of phospholipase and TPH. These results strongly suggest that analgesic mechanism by apitoxin is closely related to prostaglandins and serotonin.

  15. [Detection of Echinococcus granulosus and Echinococcus multilocularis in cyst samples using a novel single tube multiplex real-time polymerase chain reaction].

    Science.gov (United States)

    Can, Hüseyin; İnceboz, Tonay; Caner, Ayşe; Atalay Şahar, Esra; Karakavuk, Muhammet; Döşkaya, Mert; Çelebi, Fehmi; Değirmenci Döşkaya, Aysu; Gülçe İz, Sultan; Gürüz, Yüksel; Korkmaz, Metin

    2016-04-01

    Cystic echinococcosis (CE) and alveolar echinococcosis (AE) caused by Echinococcus granulosus and Echinococcus multilocularis, respectively, are important helminthic diseases worldwide as well as in our country. Epidemiological studies conducted in Turkey showed that the prevalence of CE is 291-585/100.000. It has also been showed that the seroprevalence of AE is 3.5%. For the diagnosis of CE and AE, radiological (ultrasonography, computed tomography, magnetic resonance) and serological methods, in addition to clinical findings, are being used. The definitive diagnosis relies on pathological examination When the hydatid cysts are sterile or does not contain protoscolex, problems may occur during pathological discrimination of E.granulosus and E.multilocularis species. In this study, we aimed to develop a novel multiplex real-time polymerase chain reaction (M-RT-PCR) targeting mitochondrial 12S rRNA gene of E.granulosus and E.multilocularis using Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') and Echi A (5'-GGTCTTAACTCAACTCATGGAG-3') primers and three different probes; Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fluoroscein-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-phosphate-3') and Multilocularis (5'-LC705-CTGTGATCTTGGTGTAGTAGTTGAGATT-phosphate-3') that will enable the diagnosis of CE and AE in same assay. During M-RTR-PCR, plasmids containing E.granulosus (GenBank: AF297617.1) and E.multilocularis (GenBank: NC_000928.2) mitochondrial 12S rRNA regions were used as positive controls. Cysts samples of patients which were pathologically confirmed to be CE (n: 10) and AE (n: 15) and healthy human DNA samples (n: 25) as negative control as well as DNA samples of 12 different parasites (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) were used to develop M-RT-PCR

  16. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    Science.gov (United States)

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. PMID:25479356

  17. 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.

  18. Comparison of immunocapture and RT-PCR techniques for the detection of peste-des-petits-ruminants virus (PPRV) in eye and nose swabs from infected animals

    International Nuclear Information System (INIS)

    Full text: Peste des Petits Ruminants (PPR) is a highly contagious disease of domestic and wild small ruminants. It constitutes a major constraint on production in areas where it is endemic. Classically, it is characterised by fever, nasal and ocular discharges, diarrhoea, respiratory distress, mucosal erosive lesions and death in 40-80% of acute cases. All these clinical signs, apart from the respiratory symptoms, are very similar to those of rinderpest (RP). The causal agents of both diseases are viruses which belong to the Morbillivirus genus. Described for the first time in 1942 in Cote d'Ivoire, PPR was considered for a long time as a disease of West African countries. The current knowledge on its epidemiology shows that this is no longer true, nor it is a solely African disease since it is widespread in countries lying between the Sahara and the Equator, in the Middle East and in South-West Asia. These data indicate that PPR existed undetected in most of the known endemic areas for a long time. It was overlooked because of the similarity of clinical signs to rinderpest as indicated above and also to pasteurellosis for the bronchopneumonia. The current knowledge of the disease has grown up quickly once specific diagnostic tests became available in the 1990's: serological diagnosis in the competitive format or for antigen detection by immunocapture cDNA probe and the amplification (RT-PCR) technique for the nucleic acid detection. While the immunocapture (ICE) can detect virus up to 100.6 TCID50 of virus in 50μl of sample, the limit of detection of the RT-PCR is estimated to 0.001 TCD50/ml. During a study to analyse the pathogenicity of some PPRV isolates, we have compared the efficiency of the ICE test, antibody-based antigen detection, and RT-PCR, with the RT-PCR, nucleic acid-based detection technique, to detect the shedding of the virus by the infected animals. For the study, Sahelian goats were inoculated IM with PPRV Guinea (isolate from Guinea Conakry

  19. Genome shuffling of Saccharomyces cerevisiae for enhanced glutathione yield and relative gene expression analysis using fluorescent quantitation reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Yin, Hua; Ma, Yanlin; Deng, Yang; Xu, Zhenbo; Liu, Junyan; Zhao, Junfeng; Dong, Jianjun; Yu, Junhong; Chang, Zongming

    2016-08-01

    Genome shuffling is an efficient and promising approach for the rapid improvement of microbial phenotypes. In this study, genome shuffling was applied to enhance the yield of glutathione produced by Saccharomyces cerevisiae YS86. Six isolates with subtle improvements in glutathione yield were obtained from populations generated by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant YSF2-19 strain that exhibited 3.2- and 3.3-fold increases in glutathione production in shake flask and fermenter respectively was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR (reverse transcription polymerase chain reaction). Delta CT (threshold cycle) relative quantitation analysis revealed that glutathione synthetase gene (GSH-I) expression at the transcriptional level in the YSF2-19 strain was 9.9-fold greater than in the initial YS86. The shuffled yeast strain has a potential application in brewing, other food, and pharmaceutical industries. Simultaneously, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering. PMID:27302037

  20. ESTABLISHMENT OF CRITERIA FOR MEASURING MDR-1 GENE EXPRESSION LEVEL IN BREAST CANCER BY RT-PCR

    Institute of Scientific and Technical Information of China (English)

    Liu Xiaoqing; Song Santai; Shi Chenghua; Xu Jianming; Tang Zhongming; Jiang Zefei

    1998-01-01

    Objective: To formulate criteria of multidrug resistance (mdr-1) gene expression for predicting chemotherapy response and prognosis. Methods: Using reverse transcription-polymerase chain reaction (RTPCR) assay, the expression of mdr-1 gene in 82 breast cancer samples were detected. Results: The data were treated by statistic analysis system (SAS)-singlevariate analysis. It showed that the level of mdr-1 gene expression clearly deviated from normal to right distribution (P<0.0001), and thus might be divided by quantiles P50(mdr-1/β 2-MG=0.2) and P75 (mdr-1/β 2-MG=0.6), which were taken as the preliminary criteria for analyzing 56 patients' chemosensitivity to ADM、 VDS and VCR in vitro and 32 relapsed metastatic patients' chemotherapy response in vivo, seperately. When mdr-1/β 2-MG<0.2, the ratios of resistance gradually escalated, but there were about 30%~50% of the cases who showed sensitive to the drugs in vitro and effective to chemotherapy in vivo. When mdr-1/β 2-MG≥0.6, the most of patients showed drug resistance both in vitro and in vivo. Conclusion:According to the above-mentioned results, criteria of evaluating mdr-1 gene expression level was formulated:the mdr-1/β 2-MG<0.2 (P50) was considered as negative expression, the ratio≥0.2~<0.6 (P75) was weakly positive expression, ≥0.6 was strongly positive expression. This indicated that different levels of mdr-1 gene expression may reflect objectively drug resistance in vitro and chemotherapy response in vivo.

  1. Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction.

    Science.gov (United States)

    Capote, Nieves; Gorris, M Teresa; Martínez, M Carmen; Asensio, Margarita; Olmos, Antonio; Cambra, Mariano

    2006-03-01

    ABSTRACT The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3' minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected. PMID:18944448

  2. Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem

    Directory of Open Access Journals (Sweden)

    Nina E. Nagy

    2014-04-01

    Full Text Available The tangentially oriented polyphenolic parenchyma (PP and radially organized ray parenchyma in the phloem are central in the defense of conifer stems against insects and pathogens. Laser micro-dissection enables examination of cell-specific defense responses. To examine induced defense responses in Norway spruce stems inoculated with the necrotrophic blue-stain fungus Ceratocystis polonica, RNA extracted from laser micro-dissected phloem parenchyma and vascular cambium was analyzed using real-time RT-PCR (qRT-PCR to profile transcript levels of selected resistance marker genes. The monitored transcripts included three pathogenesis-related proteins (class IV chitinase (CHI4, defensin (SPI1, peroxidase (PX3, two terpene synthesis related proteins (DXPS and LAS, one ethylene biosynthesis related protein (ACS, and a phenylalanine ammonia-lyase (PAL. Three days following inoculation, four genes (CHI4, PAL, PX3, SPI1 were differentially induced in individual cell and tissue types, both close to the inoculation site (5 mm above and, to a lesser degree, further away (10 mm above. These resistance marker genes were all highly induced in ray parenchyma, supporting the important role of the rays in spruce defense propagation. CHI4 and PAL were also induced in PP cells and in conducting secondary phloem tissues. Our data suggests that different cell types in the secondary phloem of Norway spruce have overlapping but not fully redundant roles in active host defense. Furthermore, the study demonstrates the usefulness of laser micro-dissection coupled with qRT-PCR to characterize gene expression in different cell types of conifer bark.

  3. 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.

  4. Multi-template polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Elena Kalle

    2014-12-01

    Full Text Available PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

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

    OpenAIRE

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

    2013-01-01

    Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don’t know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stabil...

  6. 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.

  7. High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

    OpenAIRE

    Moltzahn, Felix; Hunkapiller, Nathan; Mir, Alain A.; Imbar, Tal; Blelloch, Robert

    2011-01-01

    The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimi...

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

    Science.gov (United States)

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

    2015-01-01

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

  9. 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

  10. Detección y cuantificación del Potato mop-top virus (PMTV en Colombia mediante qRT-PCR

    Directory of Open Access Journals (Sweden)

    Nevar García Bastidas

    2013-04-01

    Full Text Available El Potato mop-top virus (PMTV es uno de los virus re-emergentes en cultivos de papa en Colombia. Es transmitido por Spongospora subterranea, el agente causal de la sarna polvosa. La detección del PMTV presenta dificultades debido a su distribución irregular en las plantas, bajo título y movimiento sistémico como ARN desnudo. Con el fin de ampliar el rango de herramientas disponibles para detectar el PMTV en los programas de certificación de tubérculo-semilla, en este estudio se evaluó la prueba de RT-PCR en tiempo real (qRT-PCR en dos pasos: con los cebadores PMTV-1948F/PMTV-2017R y la sonda Taqman® PMTV-1970, dirigidos al gen CP-RT del ARN2 viral. Se construyó una curva estándar a partir de la transcripción in vitro de un fragmento de 1513 pb de este gen. Posteriormente, se evaluó la utilidad de la técnica a partir de tres tipos de muestras: plantas señuelo de Nicotiana benthamiana y Solanum phureja inoculadas con quistosoros de Sss, raíces de papa con síntomas de sarna polvosa del municipio de La Unión (Antioquia y tubérculos-semilla. Mediante qRT-PCR fue posible detectar el virus en 11 de las 20 muestras de raíz de plantas señuelo, mientras que 14 de las 15 muestras de raíces de papa resultaron positivas, estimándose una concentración entre 4.72 x 10(11 y 7.60 x 10(13 partículas virales/µl. Adicionalmente, en el ensayo de tubérculo-semilla se determinó la presencia del PMTV en una de las 16 muestras. Estos resultados indican la viabilidad de utilizar rutinariamente la técnica de qRT-PCR para la detección de PMTV en Colombia.

  11. Novel molecular beacon probe-based real-time RT-PCR assay for diagnosis of Crimean-Congo hemorrhagic fever encountered in India.

    Science.gov (United States)

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

    2014-01-01

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

  12. 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...... analysis. The method was used to compare ORF 1 sequences of two divergent European-type PRRSV strains. Our results indicated that the structural and replicase parts of these two strains had evolved at overall similar rates....

  13. Influenza aviar: histopatología y detección viral por RT-PCR en tejidos fijados con formalina e incluidos en parafina

    OpenAIRE

    Roberto Padilla Noriega; Enrique Aburto Fernández; Moisés Fraire Cachón; Luis Padilla Noriega

    2004-01-01

    Se estableció la técnica de transcripción reversa-reacción en cadena de la polimerasa (RT-PCR) para la detección del virus de influenza aviar en tejidos de aves infectadas experimentalmente, incluidos en parafina, lo cual se correlacionó con las lesiones histológicas. Se inocularon por vía intravenosa ocho equilotes de diez pollos Arbor Acres de cinco semanas de edad, con 101 a 108 dosis letales de pollo del 50% (DLP50) del virus de influenza aviar A/Ck/Querétaro/20/95 (H5N2). Todas ...

  14. The Ripple Effect: Citation Chain Reactions of a Nobel Prize

    DEFF Research Database (Denmark)

    Faber Frandsen, Tove; Nicolaisen, Jeppe

    2013-01-01

    This paper explores the possible citation chain reactions of a Nobel Prize using the mathematician Robert J. Aumann as a case example. The results show that the award of the Nobel Prize in 2005 affected not only the citations to his work, but also affected the citations to the references in his...... scientific oeuvre. The results indicate that the spillover effect is almost as powerful as the effect itself. We are consequently able to document a ripple effect in which the awarding of the Nobel Prize ignites a citation chain reaction to Aumann's scientific ouvre and to the references in its nearest...

  15. Establishment of a real-time RT-PCR method based on SYBR GreenⅠfor diagnosis of porcine kobuvirus%猪嵴病毒实时荧光定量RT-PCR方法的建立

    Institute of Scientific and Technical Information of China (English)

    修金生; 陈小权; 王斌; 李涛

    2012-01-01

    A pair of specific primers targeted to the 3D gene of porcine kobuvirus was designed and a real-time reverse-transcription polymerase chain reaction (RRT-PCR) based on SYBR Green I fluorescent was developed for quantization of the porcine kobuvirus infection. The standard curve generated a wide dynamic range of 6. 42 × 102 -6. 42 × 108 DNA copies/μL with a linear correlation (R2) of 0. 999 and efficiency of 100% between the Ct value and the logarithm of the plasmid copy number. The melting curve analysis using SYBR Green I dye showed one specific peaked with a melting temperature (Tm) of (84. 94± 0. 24) ℃ with no primer-dimer peak represent. No amplificon was detected from unrelated DNA samples by this method, such as porcine transmissible gastroenteritis virus, porcine rotavirus, porcine circovirus, porcine reproductive and respiratory syndrome, pseudorabies virus, and classical swine fever virus. Kxcellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0. 26% - 1. 14% % and inter-assay of 0. 63% - 1. 79%. A real-time RT-PCR method established in this study may be used for earlier diagnosis and quantitative analysis of the infection status and the target organs infected with porcine kobuvirus.%目的 本研究根据GenBank中登录猪嵴病毒(porcine kobuvirus,PKV)株的3D蛋白序列基因特征,设计特异性引物,建立基于SYBR GreenⅠ检测PKV的实时荧光定量RT-PCR方法.结果 该方法检测PKV的3D基因在6.42×102 ~ 6.42×108拷贝/μL范围内有很好的线性关系,其扩增相关系数为0.999,扩增效率为100%,扩增产物的融解曲线分析只出现1个单特异峰,融解温度为(84.94±0.24)℃,对传染性胃肠炎病毒、猪轮状病毒、猪圆环病毒、猪繁殖与呼吸综合症病毒、猪伪狂犬病毒、猪瘟病毒均检测不到荧光信号,特异性强.所建立实时荧光定量RT-PCR方法组内变异系数为0.26%~1.14%,组间变异系数0.63%~1.79%,

  16. The generalised self-sustaining chain reaction theory about ADS

    International Nuclear Information System (INIS)

    The basic connotation of ADS (accelerator driven system) is investigated from the point of view of generalized self-sustaining chain reaction. The ADS is composed of proton accelerator, neutron producing target and the subcritical reactor. This system can be viewed entirely as a generalized self sustaining chain reaction system. In this view of point, the accelerator with target, as a part of ADS, can be viewed as an energy-neutron transformer (energy be fed to accelerator to produce medium energy protons, neutrons be produced in the heavy target as a result of proton-heavy nucleus spallation reaction). In this generalized self-sustaining chain reaction system, the number of neutrons produced after a fission event is not only the fission neutrons but also the neutrons produced by the energy-neutron transformer. That is, the ADS has more effective secondary neutrons after a fission event. It is just these additional neutrons, the ADS can be in state of self-sustaining chain reaction (criticality), although the reactor part of ADS is in state of sub-criticality. The critical equation of the generalized self-sustaining chain reaction system is presented. The relationship between the effective multiplication factors of ADS and subcritical reactor part of ADS is also presented. The power output of ADS is represented by a function of the proton current, the subcritical reactivity of the reactor in ADS, the number of neutrons produced in spallation process per proton and etc. At last, the probable application of ADS in the future is investigated

  17. Selection of suitable reference genes for quantitative RT-PCR normalization in the halophyte Halostachys caspica under salt and drought stress.

    Science.gov (United States)

    Zhang, Suwei; Zeng, Youling; Yi, Xiaoya; Zhang, Yufang

    2016-01-01

    The plants are always subjected to various environmental stress, because of plant sessile growth. qRT-PCR is a sensitive and reliable technology, and the normalization of target gene expression with suitable reference genes is very important for obtaining accurate data. Halostachys caspica is an extremely salt-tolerant halophyte belonging to Chenopodiaceae and a good candidate to explore the stress-physiological and molecular mechanism. To get truly the expression profiles of coding genes and miRNAs in H. caspica in response to salt and drought stress using qRT-PCR, suitable reference genes need to be confirmed. In this study, 10 candidate genes including ACT, UBC10, UBC13, TUB2, TUB3, EF1α, 5S rRNA, tRNA, U6 and miR1436 from H. caspica are chosen, and among them, the former nine are commonly used as internal control genes, and miR1436 with high sequence copies is no significant difference expression in high salinity-treated and untreated small RNA libraries of this species. The three softwares are used to analyze expression stability. The results showed that EF1α and TUB3 were the most stable under salt and drought stress, respectively, and UBC10 was the most constant aross all the samples with the both stressed combination. This work will benefit deep studies on abiotic tolerance in H. caspica. PMID:27527518

  18. Identification and Characterization of Lipopeptides from Bacillus subtilis B1 Against Sapstain Fungus of Rubberwood Through MALDI-TOF-MS and RT-PCR.

    Science.gov (United States)

    Sajitha, K L; Dev, Suma Arun; Maria Florence, E J

    2016-07-01

    Bacillus subtilis is a potent biocontrol agent producing a wide array of antifungal lipopeptides for the inhibition of fungal growth. B. subtilis B1 isolated from market-available compost provided an efficient control of rubberwood sapstain fungus, Lasiodiplodia theobromae. The current study is aimed to identify and characterize the lipopeptides responsible for the biocontrol of rubberwood sapstain fungus by Bacillus subtilis B1. The bacterial whole-cell surface extract from the dual culture of B. subtilis B1 and sapstain fungus (L. theobromae) was analysed using MALDI-TOF-MS. The protonated as well as sodium, potassium adducts of homologues of iturin C, surfactin, bacillomycin D and fengycin A and B were identified and expression of the lipopeptide biosynthetic genes could be confirmed through RT-PCR. This is the first report of mycobacillin and trimethylsilyl derivative of bacilysin during antagonism through MALDI-TOF-MS. MALDI-TOF-MS with RT-PCR offered easy platforms to characterize the antifungal lipopeptides. The identification of antifungal lipopeptides can lead to the formulation of prospective biocontrol by-products which have wide-scale utility. PMID:27004481

  19. Sensitive Detection of Measles Virus Infection in the Blood and Tissues of Humanized Mouse by One-step Quantitative RT-PCR

    Directory of Open Access Journals (Sweden)

    YasukoTsunetsuguYokota

    2013-10-01

    Full Text Available Live attenuated measles virus (MV has long been recognized as a safe and effective vaccine, and it has served as the basis for development of various MV-based vaccines. However, because MV is a human-tropic virus, the evaluation of MV-based vaccines has been hampered by the lack of a small-animal model. The humanized mouse, a recently developed system in which an immunodeficient mouse is transplanted with human fetal tissues or hematopoietic stem cells, may represent a suitable model. Here, we developed a sensitive one-step quantitative reverse transcription (qRT PCR that simultaneously measures nucleocapsid (N and human RNase P mRNA levels. The results can be used to monitor MV infection in a humanized mouse model. Using this method, we elucidated the replication kinetics of MV expressing EGFP both in vitro and in humanized mice in parallel with flow-cytometric analysis. Because our qRT-PCR system was sensitive enough to detect MV expression using RNA extracted from a small number of cells, it can be used to monitor MV infection in humanized mice by sequential blood sampling.

  20. 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.

  1. Detection of Listeria monocytogenes by using the polymerase chain reaction

    International Nuclear Information System (INIS)

    A method was developed for detection of Listeria monocytogens by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with 32P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains

  2. Use of polymerase chain reaction for detection of Chlamydia trachomatis

    DEFF Research Database (Denmark)

    Østergaard, Lars; Birkelund, Svend; Christiansen, Gunna

    1990-01-01

    A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. From the published sequence of the common C. trachomatis plasmid, two primer sets were selected. Detection of amplified sequences was done by agarose gel electrophoresis of cleaved or uncleaved...

  3. Characteristics of a chain thermal explosion as a function of the kinetic properties of reaction chains

    Science.gov (United States)

    Azatyan, V. V.; Piloyan, A. A.; Saikova, G. R.; Smirnov, N. N.

    2016-03-01

    Study of the combustion and explosion of hydrogen‒carbon oxide‒air mixtures shows that the sharpness of a chain thermal explosion depends on the frequency of branching in a given branch of a reaction chain. It is established that varying the CO: H2 concentration allows us to observe and eliminate the degeneration of an explosion while maintaining the regimes of ignition and deflagration.

  4. 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

  5. Establishment of Universal RT-PCR for Bovine Virus Diarrhea%牛病毒性腹泻病毒通用型RT-PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    郭春娟; 吴星星; 王璐; 季新成; 冉多良; 买买提·黑牙斯丁; 王克栋; 熊浩

    2012-01-01

    为建立一种快速、特异、简便的检测牛病毒粘膜腹泻病病毒Ⅰ、Ⅱ型抗原的通用RT-PCR方法.根据GenBank上已发表的牛病毒性粘膜腹泻病毒(BVDV)Ⅰ、Ⅱ型5’端非编码区保守序列设计合成了两条引物,其中上游引物为兼并引物.应用一步法RT-PCR技术分别对Ⅰ(Oregon C24V)、Ⅱ型(279)标准株进行扩增,将PCR产物胶回收后连与pMD18-T载体,经PCR、酶切鉴定条带均正确(288 bp),测序结果与预期一致.同时设牛疱疹病毒Ⅰ型、牛传染性鼻气管炎、猪瘟病毒、蓝舌病病毒、口蹄疫病毒、MDBK细胞作对照,结果均为阴性;并对新疆各地州的样品进行检测.优化反应条件后,检测其灵敏度为101 TCID50/mL.经对400份临床样品检测,阳性检出率为10.75%,明显高于ELISA检测.试验表明,该方法具有特异、灵敏、高效、快速、重复性好等特点,可用于牛病毒粘膜腹泻病的临床检测及流行病学检测.%To establish a rapid,specific,simple common RT-PCR method to detect bovine viral diarrhea/ mocosal disease virus type Ⅰ,Ⅱ antigen, the two synthesized primers were designed by Bovine Viral Diar-rhea/Mucosa Birus (BVDV) Ⅰ,Ⅱ type 5'Non-coding Reigion of Conserved Sequence published on Gen-Bank;one of which was an upstream and degenerate primer. I (Oregon C24V), II type (279) standard strainswere were amplified respectively by one-step RT-PCR, the PCR products gel were recovered even with the pMD18-T by PCR,restriction enzyme digestion bands were correct as 288 bp,sequencing results were in accordance with expectations. Also located bovine herpesvirus type I .Infectious Bovine Rhinotra-cheitis,classical swine fever virus,Blue tongue virus,Foot and mouth disease virus,MDBK cells were used as control,the results were negative; and also the samples from different parts of Xinjiang were tested. When reaction conditions were optimized,the sensitivity was detected as 10'TCID 50/mL. In a test of 400

  6. Diagnosis Molekuler Virus Flu Burung-A Subtipe H5 Berdasarkan Amplifikasi Gen M dan H5 dengan Metode Onestep Simplex RT-PCR (MOLECULAR DIAGNOSIS OF AVIAN INFLUENZA VIRUS TYPE A AND SUBTYPE H5 BY AMPLIFICATION OF ITS M AND H5 GENES USING ONE STEP SIMPLEX R

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2014-08-01

    Full Text Available Influenza A viruses which belong to the Family of Orthomyxoviridae are a group of viruses withsegmented ssRNA genome. The viruses can be subgroupped into many subtypes on the basis of theirsurface glycoproteins, hemagglutinin (HA and neuraminidase (NA proteins. Among the HA subtypes, H5and H7 have been found to be the most pathogenic. Conventional diagnosis of the viruses is usuallyperformed by isolation of the viruses in embryonated eggs, and hemagglutination (HA and hemagglutinationinhibition test. Although those methods are sensitive and accurate, they are time consuming and requirelaboratory facilities with high biosafety level. Commercial methods such as emzyme-linked immonosorbentassay (ELISA and immunoflurescense assay also provide a rapid result but less sensitive and specificthan conventional methods. Molecular diagnosis by amplification of M and H5 genes using one strepsimplex reverse transcriptase-polymerase chain eraction (RT-PCR provices a rapid and accurate diagnosisfor the viruses. A study was therefore conducted to evaluate the accurate and rapidity of such the moleculartests for diagnosis of avian influenza A virus, subtype H5. As many as 10 sample of the virus isolateswhich were available at the Animal Desease investigation Center in Wates, Yogyakarta, were uses in thisstudy. The virus isolates were firstly propagated in specific antigen negative (SAN chicken embryos andtested by HA/HI test. The viruses were then subjected for the RT-PCR test with varying annealingtemperatures of 500C and 520C. The result showed that all 10 isolates were type A influenza virus and 8out of 10 were influenza A subtype H5 influenza virus. RT-PCR used in this study appears to be moresensitive, rapid and accurate as compared to those by serological and isolation of the virus in embryonatedeggs.

  7. Establishment of duplex RT-PCR detection for foot and mouth disease virus and bovine enterovirus%同时检测口蹄疫病毒和牛肠道病毒双重RT-PCR方法的建立

    Institute of Scientific and Technical Information of China (English)

    吴丹; 吴涛; 张鹤晓; 高志强; 蒲静; 汪琳; 乔彩霞; 谷强; 夏春

    2012-01-01

    To establish a rapid method for detection of foot and mouth disease virus (FMDV) and bovine enterovirus (BEV) infections, a duplex RT-PCR assay was developed with 2 pairs of primers for 5'UTR sequences of FMDV and BEV. Under the optimized conditions, the assay was specifically detection of FMDV and BEV, and no cross-reactions with other related bovine viruses. In addition, the detection limit of this method was 8 TC1D5()/0.l mL of FMDV and 2 TCHV0.1 mL of BEV. Furthermore, tested on 852 clinical samples collected from Beijing areas, the results showed that 18 samples were positive for FMDV, 117 samples were positive for BEV, and 10 samples were positive for both, which were completely consistent with the results by virus isolation. Our data showed that the duplex RT-PCR assay established in the study was rapid, specificity, high sensitivity and repeatability, which provides a useful technical support for detection of FMDV and BEV.%为建立同时检测口蹄疫病毒(FMDV)和牛肠道病毒(BEV)的双重RT-PCR方法,本研究根据已发表的FMDV和BEV基因的5'UTR保守区域,分别设计、筛选出一对特异性引物建立了双重RT-PCR方法,并优化了反应体系和条件.采用该方法检测其他几种牛相关病毒结果均为阴性,表明特异性良好.该方法对FMDV的最低检出限为8 TCID50/0.1 mL,对BEV的最低检出限为2 TCID50/0.1 mL,表明其具有良好的敏感性.采用该方法检测了852份临床样品,并与病毒分离方法比较,结果两种方法检测结果完全一致.本研究首次建立了同时检测FMDV和BEV的双重RT-PCR快速检测方法,该方法的建立为FMDV和BEV的检测提供了一种技术手段.

  8. Development of quantitative RT-PCR assays for wild-type urokinase receptor (uPAR-wt) and its splice variant uPAR-del5

    International Nuclear Information System (INIS)

    The receptor for the serine protease urokinase-type plasminogen activator, uPAR (CD 87), plays an important role in tumor cell invasion and metastasis of solid malignant tumors. uPAR is a highly glycosylated, glycan lipid-anchored membrane protein, consisting of three homologous domains. Each individual domain is encoded by two exons: DI by exons 2+3, DII by exons 4+5, and DIII by exons 6+7. Beside the wild-type (wt) uPAR mRNA, two splice variants either lacking exon 5 (uPAR-del5) or both exons 4 and 5 (uPARdel4/ 5) have been described. Previously, we studied expression of the mRNA variant uPAR-del4/5 and uPAR mRNA encompassing exons 2, 3, and 4 (i.e. uPAR-wt plus uPAR-del5) applying real-time RT-PCR assays for quantification of the mRNA concentration. In the present paper, we established two additional specific, robust and highly sensitive RT-PCR assays, based on the LightCycler technology, to specifically quantify either uPAR-wt or its splice variant, uPARdel5. Expression of uPAR-wt and uPAR-del5 was analyzed in different human malignant cell lines (ovarian cancer cell lines OVMZ-6 and OVMZ-10; breast cancer cell lines MDA-MB 231, MDA-MB 231 BAG, MDA-MB 435, and aMCF-7; brain tumor cell line LN 18) as well as in a set of 174 breast cancer tissue samples. uPAR-del5 mRNA was found to be expressed very frequently at a rather low level (typically less than 1% of uPAR-wt mRNA). In tumor tissue from breast cancer patients, a statistically significant correlation between uPAR-del5 and uPAR-wt mRNA (r = 0.779; P < 0.001) was observed. There was no association between the expression level of either mRNA and clinical parameters such as nodal status, tumor size and grade. In estrogen receptor negative tumors, a significantly higher uPAR-del5 expression was found (P 0.023). The two developed quantitative RT-PCR assays described here may aid further analysis of the function and clinical relevance of uPAR-wt and one of its splice variants, uPAR-del5, in malignant tumors

  9. Possibility for use of RT-PCR technique in establishing presence of bovine viral diarrhea virus in sperm of breeding bulls

    Directory of Open Access Journals (Sweden)

    Petrović Tamaš

    2005-01-01

    Full Text Available The bovine viral diarrhea (BVD virus is a significant health-economic pathogen in cattle which can be excreted and spread also through sperm of persistently or acutely infected bulls. Native sperm of 6 bulls, found to be negative to the BVD virus by isolating the virus and using the RT-PCR method, was experimentally infected with a tenfold dilution of the non-cytopathogen 22146 strain of the BVD virus with a titer of 105,5. This way, dilutions of the BVD virus from 10-1 to 10-6 (5 x 104 TCID/50 do 0,5 TCID/50 in 0.1 ml native sperm were obtained. From sperm infected in this way, the virus was reisolated on FTB cell culture in a microtiter plate with 96 pools in which each sample of the infected sperm was set up in three samples, and each of them was titrated to a dilution of 1:2 to 1:256. The presence of the BVD virus was proven using the technique of fluorescent antibodies in a second blind passage on FTB culture cells. For cell culture an extremely toxic effect of native sperm to a dilution of 1:64 was established. The BVD virus was reisolated from sperm in all three sperm samples with 5 x 104, 5 x 103 i 5 x 102 TCID/50, and it was not reisolated from sperm with 50, with 5, and with 0.5 TCID/50 BVD virus in 0.1 ml native sperm. At the same time, the presence of the BVD viral genome was proved using the RT-PCR method in the same samples of artificially infected native sperm of bulls. A positive re suit was established in native sperm with 5 x 104, 5 x 103, 5 x 102 and 50 TCID/50 BVD virus n 0.1 ml native sperm. The experiment proved that the RT-PCR method has advantages over the isolation of the BVD virus from samples of native sperm of bulls. These are: shortterm investigations (1 to 2 days and greater sensitivity (10 times bigger than the isolation of the virus. The isolation of the virus takes at least 10 days, and its greater sensitivity is primarily a result of the cyrotoxic effect of native sperm of bulls on cell culture.

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

    Science.gov (United States)

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

    2010-08-01

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

  11. Influenza aviar: histopatología y detección viral por RT-PCR en tejidos fijados con formalina e incluidos en parafina

    Directory of Open Access Journals (Sweden)

    Roberto Padilla Noriega

    2004-01-01

    Full Text Available Se estableció la técnica de transcripción reversa-reacción en cadena de la polimerasa (RT-PCR para la detección del virus de influenza aviar en tejidos de aves infectadas experimentalmente, incluidos en parafina, lo cual se correlacionó con las lesiones histológicas. Se inocularon por vía intravenosa ocho equilotes de diez pollos Arbor Acres de cinco semanas de edad, con 101 a 108 dosis letales de pollo del 50% (DLP50 del virus de influenza aviar A/Ck/Querétaro/20/95 (H5N2. Todas las aves se sometieron a evaluación histopatológica. Se ensayaron por RT-PCR tres tipos de tejidos incluidos en parafina (un ave por dosis: pulmón, encéfalo y riñón. Las principales lesiones histológicas observadas en las aves infectadas incluyeron necrosis del tejido linfoide (en pulmón, bazo, bolsa de Fabricio y tonsilas cecales, vasculitis, necrosis tubular aguda, encefalitis no supurativa y necrosis multifocal en los órganos examinados. Las principales diferencias con otras cepas altamente patogénicas ?peste de las aves (H7N7, Ck/Scot/59 (H5N1, Tern/SA (H5N3, Ty/Ont/66 (H5N9 y Ck/Penn/83 (H5N2? fueron: mayor frecuencia de daño vascular, nefropatogenicidad, necrosis en islotes pancreáticos y necrosis difusa del tejido linfoide. Estos hallazgos le confieren a la cepa bajo estudio características propias distintivas. Se detectó virus de influenza en pulmón con la técnica de RT-PCR, independientemente de la dosis, y en encéfalo y riñón en siete de las ocho dosis empleadas, lo cual indica que el virus se replica en estos tejidos; los cambios patológicos observados pueden atribuirse a la replicación viral, sin excluir la participación de otros mecanismos de daño celular.

  12. Identification of MicroRNAs in Response to Different Day Lengths in Soybean Using High-Throughput Sequencing and qRT-PCR.

    Directory of Open Access Journals (Sweden)

    Wenbin Li

    Full Text Available MicroRNAs (miRNAs are short, non-coding single-strand RNA molecules that play important roles in plant growth, development and stress responses. Flowering time affects the seed yield and quality of soybean. However, the miRNAs involved in the regulation of flowering time in soybean have not been reported until recently. Here, high-throughput sequencing and qRT-PCR were used to identify miRNAs involved in soybean photoperiodic pathways. The first trifoliate leaves of soybean that receive the signal of light treatment were used to construct six libraries (0, 8, and 16 h under short-day (SD treatment and 0, 8, and 16 h under long-day (LD treatment. The libraries were sequenced using Illumina Solexa. A total of 318 known plant miRNAs belonging to 163 miRNA families and 81 novel predicted miRNAs were identified. Among these, 23 miRNAs at 0 h, 65 miRNAs at 8 h and 83 miRNAs at 16 h, including six novel predicted miRNAs at 8 h and six novel predicted miRNAs at 16 h, showed differences in abundance between LD and SD treatments. Furthermore, the results of GO and KEGG analyses indicated that most of the miRNA targets were transcription factors. Seven miRNAs at 0 h, 23 miRNAs (including four novel predicted miRNAs at 8 h, 16 miRNAs (including one novel predicted miRNA at 16 h and miRNA targets were selected for qRT-PCR analysis to assess the accuracy of the sequencing and target prediction. The results indicated that the expression patterns of the selected miRNAs and miRNA targets showed no differences between the qRT-PCR and sequencing results. In addition, 23 miRNAs at 0 h, 65 miRNAs at 8 h and 83 miRNAs at 16 h responded to day length changes in soybean, including six novel predicted miRNAs at 8 h and six novel predicted miRNAs at 16 h. These results provided an important molecular basis to understand the regulation of flowering time through photoperiodic pathways in soybean.

  13. Quantitative RT-PCR Comparison of the Urea and Nitric Oxide Cycle Gene Transcripts in Adult Human Tissues

    OpenAIRE

    Neill, Meaghan Anne; Aschner, Judy; Barr, Frederick; Summar, Marshall L.

    2009-01-01

    The urea cycle and nitric oxide cycle play significant roles in complex biochemical and physiologic reactions. These cycles have distinct biochemical goals including the clearance of waste nitrogen; the production of the intermediates ornithine, citrulline, and arginine for the urea cycle; and the production of nitric oxide for the nitric oxide pathway. Despite their disparate functions, the two pathways share two enzymes, argininosuccinic acid synthase and argininosuccinic acid lyase, and a ...

  14. Detection of Neisseria Gonorrhoeae from Urine with Ligase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    曹经江; 郑和义; 胡维

    2003-01-01

    Objective: To evaluate the value of ligase chain reaction(LCR) in the diagnosis of diplococcus gonorrhoeae in urine.Methods: LCR detection of the urine for Neisseria gonorrhoeae and bacteria culture of discharge was per-formed simultaneously to 276 patients with urethritis or cervicitis seeking treatment in sex transmitted dis-eases (STDs) outpatient clinic. For specimens with discordant results, polymerase chain reaction was conducted. The purpose was to detect the respective sensitivity and specificity of bacteria culture and LCR. Results: 24 of 276(8.7%) patients had positive LCR results and 21 of 276(7.6%) were positive for culture.5 specimens had discordant results from LCR and bacteria culture. The sensitivity and specificity of LCR in the diagnosis of gonorrhoeae were 92.3% and 100% respectively. Conclusion: This study showed that LCR had a higher sensitivity and specificity for the diagnosis of gonorrhoeae from urine.

  15. The chain gas phase reactions. The present problems

    International Nuclear Information System (INIS)

    The results of investigations of chain gas phase reactions which are directed for solving practically important problems have been carried out. The problem of increasing methanol formation selectivity in the direct natural gas oxidation process is discussed. The peculiarities of cool flame of cyclic hydrocarbons, particularly cyclohexane which are containing in the different kinds of fuel. The influence of cool flame on intensity and full consumption of fuels burning is examined. Conjugated processes of SO2 conversion to SO3 and sulfur under effect of hydrocarbon and hydrogen oxidation chain gas phase reactions are considered. The results of discussed investigations can be served as a basis for the developing of industrial processes for conversion natural hydrocarbon row materials and also the ecological problems of utilization of SO2 gas ejected from thermoelectric power stations and metallurgical plants

  16. Amplification and Characterization of Bull Semen Infected Naturally with Foot-and-mouth Disease Virus Type Asial by RT-PCR

    Institute of Scientific and Technical Information of China (English)

    Jun-jun SHAO; Xiang-tao LIU; Zai-xin LIU; Ji-xing LIU; Hui-yun CHANG; Tong LIN; Guo-zheng CONG; Jun-zheng DU; Jian-hong GUO; Hui-fang BAO; You-jun SHANG; Ya-min YANG

    2008-01-01

    To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group.

  17. Detection and confirmation of PPR virus antigen in sheep and goats by sandwich-ELISA and RT-PCR in Andhra Pradesh, India

    Directory of Open Access Journals (Sweden)

    G. Saritha

    2015-06-01

    Full Text Available Peste des petits ruminants (PPR is a highly contagious disease of domestic and wild small ruminants. Rapid and accurate laboratory assay are essential to enable the implementation of appropriate control strategies to restrict the spread of PPR. The present study was designed to detect the PPR virus (PPRV antigen (N-gene in nasal swabs and tissue samples. A total of 195 samples comprising of 138 nasal swabs from PPR suspected sheep (n=72 and goats (n=66, and 57 tissue samples comprising of lymph nodes from dead sheep (n=39 and goats (n=18 were collected from certain parts of Andhra Pradesh. The samples were subjected to sandwich-ELISA followed by RT-PCR for confirmatory diagnosis. In this study, PPRV could be detected in 27.53% (n=38/138 nasal swabs and 49.12% (n=28/57 tissue samples. Data showed that PPRV infection is widespread in the Andhra Pradesh, India.

  18. Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development

    DEFF Research Database (Denmark)

    Kaczmarczyk, Agnieszka Ewa; Bowra, Steve; Elek, Zoltan;

    2012-01-01

    Background Cereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene...... expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging. Results We designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and gamma......-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families...

  19. Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus

    DEFF Research Database (Denmark)

    Leblanc, N; Rasmussen, Thomas Bruun; Fernandez, J;

    2010-01-01

    A real-time RT-PCR assay based on the primer–probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward...... tests showed no positive results for heterologous pathogens. The assay was tested on clinical samples from BTV 8 outbreaks in Sweden and Denmark in 2008. The lowest detection limit for that serotype, determined with PCR standards, was 57 genome copies. The assay sensitivity for some other serotypes that...... circulate currently in Europe was also determined. BTV 2, 4, 9 and 16 were tested on available cell culture samples and the detection limits were 109, 12, 13 and 24 copies, respectively. This assay provides an important tool for early and rapid detection of a wide range of BTV strains, including emerging...

  20. 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; Kusk, M.; Zhang, M.F.; Jorgensen, P.H.

    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...... of a suitable internal control gene, real time PCR parameters were evaluated for three candidate genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA and beta-actin to IBDVs. Based on this P-actin was selected as an internal control for quantification of IBDVs in BF. All BF samples with...... primers. The method described here is robust and may sever as a useful tool with high capacity for diagnostics as well as in viral pathogenesis studies....

  1. Polymerase chain reaction: A molecular diagnostic tool in periodontology

    OpenAIRE

    Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

    2016-01-01

    This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology w...

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

    Science.gov (United States)

    Abdelmigid, Hala M

    2016-03-01

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

  3. Improvement of the clinical applicability of the Genomic Grade Index through a qRT-PCR test performed on frozen and formalin-fixed paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Piccart Martine

    2009-09-01

    Full Text Available Abstract Background Proliferation and tumor differentiation captured by the genomic grade index (GGI are important prognostic indicators in breast cancer (BC especially for the estrogen receptor positive (ER+ disease. The aims of this study were to convert this microarray index to a qRT-PCR assay (PCR-GGI, which could be realized on formalin fixed paraffin embedded samples (FFPE, and to assess its prognostic performance and predictive value of clinical benefit in early and advanced ER+ BC patients treated with tamoxifen. Methods The accuracy and concordance of the PCR-GGI with the GGI was assessed using BC patients for which frozen and FFPE tissues as well as microarray data were available (n = 19. The evaluation of the prognostic value of the PCR-GGI was assessed on FFPE material using a consecutive series of 212 systemically treated early BC patients. The predictive performance for tamoxifen benefit was assessed using two ER+ BC populations treated either with adjuvant tamoxifen only (n = 77+139 or first-line tamoxifen for advanced disease (n = 270. Results The PCR-GGI is based on the expression of 8 genes (4 representative of the GGI and 4 reference genes. A significant correlation was observed between the microarray-derived GGI and the qRT-PCR assay using frozen (ρ = 0.95, p univar. = 1.89; [95CI:1.01-3.54], p = 0.05. The PCR-GGI further identified two subgroups of patients with statistically different time to distant metastasis free survival (DMFS across the two cohorts of ER+ BC patients treated with adjuvant tamoxifen. Additionally, the PCR-GGI was associated with response to tamoxifen in the advanced setting (HRunivar. = 1.98; [95CI:1.51-2.59], p = 6.9E-07. Conclusion PCR-GGI recapitulates in an accurate and reproducible manner the performances of the GGI using frozen and FFPE samples.

  4. Establishment and Application of RT-PCR Detection of Pocrine Kobuvirus%猪嵴病毒RT-PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    张文波; 冷闯; 段俊; 邓舜洲

    2013-01-01

    A detection method for porcine kobuvirus (PKV) with RT-PCR technique was established to detect the 3D gene of PKV from the intestinal tract of diarrhea piglets, with the detection limit about 2. 3 pg RNA. 50 samples of diarrhea piglets were detected by the RT-PCR method, the PKV positive rate of samples was 58%. The PCR detection method established in this study had the characteristics of sensitivity, high specificity and good repeatability, and could be used in molecular epidemi-ological investigation and rapid clinical diagnosis of PKV.%根据GenBank中登录的猪嵴病毒3D基因保守序列,设计合成1对引物,以腹泻哺乳仔猪的小肠为起始材料,建立了猪峭病毒的RT-PCR检测方法,并用该方法对收集的50份哺乳仔猪腹泻样本进行检测.结果显示,该方法对模板的最小检测量是2.3 pg,检测猪其他常见致腹泻病毒均为阴性,同一样品3次试验结果一致;所扩增的序列经测序分析均为猪嵴病毒的3D核苷酸序列;仔猪腹泻样本的猪嵴病毒检出率达58%.结果表明,该方法敏感度高、重复性和特异性良好,可用于猪嵴病毒的分子流行病学调查和临床病例的快速诊断.

  5. 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. PMID:24466312

  6. Reaction chain modeling of denitrification reactions during a push-pull test

    Science.gov (United States)

    Boisson, A.; de Anna, P.; Bour, O.; Le Borgne, T.; Labasque, T.; Aquilina, L.

    2013-05-01

    Field quantitative estimation of reaction kinetics is required to enhance our understanding of biogeochemical reactions in aquifers. We extended the analytical solution developed by Haggerty et al. (1998) to model an entire 1st order reaction chain and estimate the kinetic parameters for each reaction step of the denitrification process. We then assessed the ability of this reaction chain to model biogeochemical reactions by comparing it with experimental results from a push-pull test in a fractured crystalline aquifer (Ploemeur, French Brittany). Nitrates were used as the reactive tracer, since denitrification involves the sequential reduction of nitrates to nitrogen gas through a chain reaction (NO3- → NO2- → NO → N2O → N2) under anaerobic conditions. The kinetics of nitrate consumption and by-product formation (NO2-, N2O) during autotrophic denitrification were quantified by using a reactive tracer (NO3-) and a non-reactive tracer (Br-). The formation of reaction by-products (NO2-, N2O, N2) has not been previously considered using a reaction chain approach. Comparison of Br- and NO3- breakthrough curves showed that 10% of the injected NO3- molar mass was transformed during the 12 h experiment (2% into NO2-, 1% into N2O and the rest into N2 and NO). Similar results, but with slower kinetics, were obtained from laboratory experiments in reactors. The good agreement between the model and the field data shows that the complete denitrification process can be efficiently modeled as a sequence of first order reactions. The 1st order kinetics coefficients obtained through modeling were as follows: k1 = 0.023 h- 1, k2 = 0.59 h- 1, k3 = 16 h- 1, and k4 = 5.5 h- 1. A next step will be to assess the variability of field reactivity using the methodology developed for modeling push-pull tracer tests.

  7. Radioinitiation of Chain Branched Reactions and its Sensitization

    International Nuclear Information System (INIS)

    This paper describes the results of experiments by the writers with radioinitiation of chain branched reactions of the oxidation of organic compounds. The function of radiation as an initiating agent is described with reference to the oxidation of several unsaturated hydrocarbons and butanol. The reaction is self-accelerating and proceeds spontaneously after radiation has ceased. A detailed investigation was made of a process from oxidizing benzene, which has a high radiation resistance. The writers devised a method of sensitizing the radioinitiation of the oxidation of radiation-resistant substances by chemically inert but non-radiation-resistant substances. The main quantitative features of the process for the radiooxidation of benzene are stated to be the accumulation of various reaction products, and the effect of temperature, pressure, power and radiation dosage on the process of such accumulation. Information was obtained about the mechanism of the process. The design of circulating equipment is described. (author)

  8. Convective polymerase chain reaction around micro immersion heater

    Science.gov (United States)

    Hennig, Martin; Braun, Dieter

    2005-10-01

    Polymerase chain reaction (PCR) is performed in the thermal convection created by a micro immersion heater. Instead of repetitive heating and cooling, the temperature gradient induces thermal convection which drives the reaction liquid between hot and cold parts of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates with the use of proteins into twice the amount in the cold region. The constant heater is simply dipped into the reaction solution. Compared to previous experiments, we demonstrate that convective PCR is possible in a robotically accessible open vessel. Our approach compares well with fast PCR cyclers and replicates DNA 500 000 fold within 20minutes. We reduce the necessary components for PCR to cheap, single-use components and therefore increasing the prospects of bringing PCR to point of care applications—even in third world countries.

  9. 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

  10. Application of polymerase chain reaction to detect rearrangement of immunoglobulin heavy chain genes in lymphoproliferative disease.

    Science.gov (United States)

    Khalil, S H; Siegrist, K; Akhtar, M

    1997-07-01

    As part of our routine work-up in the diagnosis of lymphoproliferative disease, we used a rapid polymerase chain reaction (PCR) assay to amplify the DNA fragments of the framework 3 (FR3) region of the immunoglobulin heavy (IgH) chain genes. The assay does not involve hybridization, nested priming, or sequencing of the amplified PCR product. It was performed on 66 specimens of B-cell lymphoproliferative disease, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, hairy cell leukemia and follicular lymphoma. Twenty-six specimens of negative controls, including acute myeloid leukemia, chronic myeloid leukemia in myeloid transformation and idiopathic thrombocytopenic purpura, were also analyzed. The assay was performed with 77% sensitivity and 100% specificity. The standard IgH chain gene rearrangement by Southern blot analysis is reserved for the remaining negative cases if clinically indicated. PMID:17353588

  11. 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. PMID:25728411

  12. Comparision of clinical diagnostic value between PCR and TaqMan RT-PCR for Mycoplasma pneumoniae in throat swabs%PCR及RT-PCR检测咽拭子标本肺炎支原体的比较

    Institute of Scientific and Technical Information of China (English)

    李淳; 吴移谋; 朱翠明; 钟礼立; 陈丹; 吕建华

    2012-01-01

    To compare the clinical diagnostic value between PCR and RT PCR for M. Pneumoniae in swab, we per formed both PCR and RT PCR assays analysis for M. Pneumoniae DNA on a total of 566 samples of throat swab from 106 ped iatric children with M. Pneumoniae and in whom M. Pneumoniae was suspected. Among the 566 pediatric children, there were 45(7. 95%) PCR positive specimens and 175(30. 92%) RT PCR positive specimens. In the 106 pediatric children with M. Pneumoniae, 5 were positive for PCR, and 95 were positive for RT PCR. In the 460 pediatric children with symptom of M. Pneumoniae, 40 were positive for PCR, and 80 were positive for RT PCR. The sensitivy of Rt PCR for M. Pneumoniae detec tion appeared to be better than that of PCR. (sensitivity . RT PCR 89. 62 % , PCR 4. 72 % , x2=146. 322, P = 0. 000), but there was no significant difference in the specificity between RT PCR and PCR (specifitivity: RT PCR 82. 60 %, PCR 91. 30%,x2 - 3. 331, P - 0. 068). It is concluded that the TaqMan based RT PCR assay is a rapid, sensitive and specific meth od for the detection of M. Pneumoniae in throat swabs of children in early period of diagnosis.%目的 应用聚合酶链反应(PCR)与实时taqMan荧光定量PCR(RT-PCR)检测咽拭子标本中的肺炎支原体DNA(Mp-DNA),比较2种方法检测结果的临床诊断价值.方法 随机选取I临床儿科门诊患儿566例,包括临床治诊Mp感染患儿106例和临床疑似Mp感染忠儿460例,分别采用PCR法和RT-PCR法检测,以临床治诊Mp作为参照标准,采用x2检验评定2种检测方法诊断的灵敏度和特异度,比较2种检测方法对Mp的诊断价值.结果 566份受检患儿的咽拭子标本中,PCR法检测阳性45例(7.95%)(临床治诊Mp感染患儿5例,临床疑似Mp感染患儿40例),RT-PCR法检测阳性175例(30.92%)(临床治诊Mp感染患儿95例,临床疑似Mp感染患儿80例).RT-PCR法检测咽拭子Mp-DNA诊断Mp感染的敏感度显著高于PCR法(敏感度RT-PCR 89.62%,PCR 4.72%,x2=146.322,P

  13. 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.

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

    OpenAIRE

    Alves-Ferreira Marcio; Grossi-de-Sa Maria; Brilhante Osmundo; Nardeli Sarah M; Artico Sinara

    2010-01-01

    Abstract Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on i...

  15. Robust quantification of polymerase chain reactions using global fitting.

    Directory of Open Access Journals (Sweden)

    Ana C Carr

    Full Text Available BACKGROUND: Quantitative polymerase chain reactions (qPCR are used to monitor relative changes in very small amounts of DNA. One drawback to qPCR is reproducibility: measuring the same sample multiple times can yield data that is so noisy that important differences can be dismissed. Numerous analytical methods have been employed that can extract the relative template abundance between samples. However, each method is sensitive to baseline assignment and to the unique shape profiles of individual reactions, which gives rise to increased variance stemming from the analytical procedure itself. PRINCIPAL FINDINGS: We developed a simple mathematical model that accurately describes the entire PCR reaction profile using only two reaction variables that depict the maximum capacity of the reaction and feedback inhibition. This model allows quantification that is more accurate than existing methods and takes advantage of the brighter fluorescence signals from later cycles. Because the model describes the entire reaction, the influences of baseline adjustment errors, reaction efficiencies, template abundance, and signal loss per cycle could be formalized. We determined that the common cycle-threshold method of data analysis introduces unnecessary variance because of inappropriate baseline adjustments, a dynamic reaction efficiency, and also a reliance on data with a low signal-to-noise ratio. SIGNIFICANCE: Using our model, fits to raw data can be used to determine template abundance with high precision, even when the data contains baseline and signal loss defects. This improvement reduces the time and cost associated with qPCR and should be applicable in a variety of academic, clinical, and biotechnological settings.

  16. Implosion chain reaction mitigation in underwater assemblies of photomultiplier tubes

    International Nuclear Information System (INIS)

    Since the accident with a cascade failure of photomultiplier tubes (PMTs) in the Super-Kamiokande experiment in 2001, the mechanical performance of large format semi-hemispherical PMTs has become a critical issue for large water Cherenkov detectors. The subject of this study is the survival of an assembled array of PMTs under significant hydrostatic pressure and subjected to shock waves caused by the failure of a single PMT. This paper details the results of the second stage of a R and D program focused on the design and testing of different PMT assemblies to mitigate the risk of a “chain-reaction” of PMT failures. The initial results show that our PMT assembly design can effectively reduce the magnitude of the shock wave. With the testing results in this paper and the hydrodynamic simulation calculation, we can further improve the design of PMT deployment to mitigate the risk of chain reactions caused by implosion induced shock waves

  17. Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique

    OpenAIRE

    İLHAK, O. İrfan; Arslan, Ali

    2007-01-01

    The origin of horse, dog, cat, bovine, sheep, porcine, and goat meat was determined by the polymerase chain reaction (PCR) technique, using species-specific primers. Test mixtures of meat were prepared by adding 5%, 2.5%, 1%, 0.5%, and 0.1% levels of pork, horse, cat, or dog meat to beef, sheep, and goat meat. Samples taken from those combinations were analyzed by PCR for species determination. Mitochondrial DNA (mt DNA) fragments of 439, 322, 274, 271, 225, 212, and 157 bp for horse, dog, ca...

  18. Identification of Erwinia stewartii by a ligase chain reaction assay.

    OpenAIRE

    Wilson, W.J.; Wiedmann, M; Dillard, H. R.; Batt, C A

    1994-01-01

    A PCR-coupled ligase chain reaction (LCR) assay was developed to distinguish the plant pathogenic bacterium Erwinia stewartii from other erwiniae. This new technique allows discrimination to the species level on the basis of a single-base-pair difference in the 16S rRNA gene which is unique to E. stewartii. Portions of the 16S rRNA genes of E. stewartii and the closely related Erwinia herbicola were sequenced. From comparison of the two 16S rRNA gene regions, two primer pairs were constructed...

  19. A chain reaction approach to modelling gene pathways.

    Science.gov (United States)

    Cheng, Gary C; Chen, Dung-Tsa; Chen, James J; Soong, Seng-Jaw; Lamartiniere, Coral; Barnes, Stephen

    2012-08-01

    BACKGROUND: Of great interest in cancer prevention is how nutrient components affect gene pathways associated with the physiological events of puberty. Nutrient-gene interactions may cause changes in breast or prostate cells and, therefore, may result in cancer risk later in life. Analysis of gene pathways can lead to insights about nutrient-gene interactions and the development of more effective prevention approaches to reduce cancer risk. To date, researchers have relied heavily upon experimental assays (such as microarray analysis, etc.) to identify genes and their associated pathways that are affected by nutrient and diets. However, the vast number of genes and combinations of gene pathways, coupled with the expense of the experimental analyses, has delayed the progress of gene-pathway research. The development of an analytical approach based on available test data could greatly benefit the evaluation of gene pathways, and thus advance the study of nutrient-gene interactions in cancer prevention. In the present study, we have proposed a chain reaction model to simulate gene pathways, in which the gene expression changes through the pathway are represented by the species undergoing a set of chemical reactions. We have also developed a numerical tool to solve for the species changes due to the chain reactions over time. Through this approach we can examine the impact of nutrient-containing diets on the gene pathway; moreover, transformation of genes over time with a nutrient treatment can be observed numerically, which is very difficult to achieve experimentally. We apply this approach to microarray analysis data from an experiment which involved the effects of three polyphenols (nutrient treatments), epigallo-catechin-3-O-gallate (EGCG), genistein, and resveratrol, in a study of nutrient-gene interaction in the estrogen synthesis pathway during puberty. RESULTS: In this preliminary study, the estrogen synthesis pathway was simulated by a chain reaction model. By

  20. Comparison of different protocols for the bovine viral diarrhea virus detection by RT-PCR in pools of whole blood and blood serum artificially contaminated/ Comparação de diferentes protocolos para a detecção do vírus da diarréia viral bovina por RT-PCR em grupos de sangue total e de soro sangüíneo, artificialmente contaminados

    Directory of Open Access Journals (Sweden)

    Amauri A. Alfieri

    2005-06-01

    Full Text Available The RT-PCR technique was optimized and evaluated to detect the 5’ untranslated region of bovine viral diarrhea virus (BVDV from clinical samples that consisted of blood serum and whole blood artificially contaminated with the NADL strain of BVDV. To optimization of technique, the following conditions were evaluated: i two pairs of primers, 103 / 372 (WEINSTOCK et al., 2001 and 324 / 326 (VILCEK et al., 1994, ii four methods of nucleic acid extraction (phenol/chloroform/isoamyl alcohol; silica/guanidine isothiocyanate; a combination of the two previous methods; and TRIzol™ and iii different concentrations and compositions of reagents and time/temperature of the reactions. Between the alternatives tested that resulted in the amplification of the 290 bp product that was easily visualized in ethidium bromide stained 2% agarose gel was that presented the following conditions: i primers 103 and 372; ii initial volume and clinical sample: 50 mL of blood serum; iii extraction of nucleic acid: silica/guanidine isothiocyanate method; iv reverse transcription: 9 mL extracted nucleic acid, 1xPCR buffer (20 mM Tris-HCl pH 8.4 and 50 mM KCl, 1.5 mM MgCl2; 60 units of reverse transcriptase enzyme M-MLV, RNA denaturation at 97°C / 4 min, and reverse transcription at 42°C / 30 min; v PCR: primers 103 / 372 with anneling temperature at 59°C. The utilization of RT-PCR within these optimized conditions allowed the amplification of the BVDV NADL strain (103,56 TCID50 from pools of artificially contaminated blood serum until the dilution 1:160.A técnica da RT-PCR foi otimizada e avaliada para a detecção da região 5’ terminal não-traduzida do genoma do vírus da diarréia viral bovina (BVDV em amostras clínicas de bovinos, constituídas por soro sangüíneo e sangue total, artificialmente contaminadas com a estirpe NADL do BVDV. Para a otimização da técnica foram avaliados: i dois pares de primers, 103 / 372 (WEINSTOCK et al., 2001 e 324 / 326

  1. Virus isolation and molecular characterization of canine distemper virus by RT-PCR from a mature dog with multifocal encephalomyelit Isolamento e caracterização molecular do vírus da cinomose canina por RT-PCR a partir de um cão adulto com encefalomielite multifocal

    Directory of Open Access Journals (Sweden)

    Alexandre Mendes Amude

    2007-06-01

    Full Text Available A case of multifocal distemper encephalomyelitis in a mature dog is described. In the presented case the ante mortem clinical diagnosis of canine distemper virus (CDV infection could not be ideally performed due to the absence of typical signs of distemper, such as myoclonus and systemic signs accompanying the nervous signs. The definitive diagnosis of distemper encephalomyelitis was only carried out at post mortem through virus isolation in cell culture from fresh central nervous system (CNS fragments and CDV nucleoprotein gene detection in the CNS by RT-PCR.Descreve-se um caso de encefalomielite multifocal pela cinomose em um cão adulto. No caso apresentado o diagnóstico clínico da infecção pelo vírus da cinomose canina (CDV não pode ser adequadamente realizado devido à ausência de sinais típicos da enfermidade, tais como mioclonia e sinais sistêmicos. O diagnóstico definitivo somente foi possível post mortem pelo isolamento do CDV em cultivo celular a partir dos fragmentos frescos do sistema nervoso central (SNC e pela detecção do gene da nucleoproteína do CDV em fragmentos do SNC por meio da RT-PCR.

  2. APPLICATION OF POLYMERASE CHAIN REACTION FOR DIAGNOSING AMEBIC LIVER ABSCESS

    Institute of Scientific and Technical Information of China (English)

    郭增柱; 王正仪; 安亦军; 祝宏

    1996-01-01

    Polymerase chain reaction (PCR) has been applied in diagnosing amebic liver infection by detecting pathogenic Entamoeba histolytica DNA in liver aspirates. Oligonucleotide primers found to he specific for the gene encoding the 30 kDa molecule of this pathogenic ameba were used in the test. Liver aspirates obtained from 23 patients with amebic liver abscess substantiated by typical clinical rnanifastation or with very high titres of anti-E histtolytica antibodies by ELISA were found to he positive by PCR. Fourteen controlsamples (3 cases of bacterial liver abscess, I of liver cancer and 10 of other abscess) were all found to be negative to this reaction. The results suggested PCR to he a specific and sensitive tool for diagnosing amebic liver abscess infections.

  3. Electrochemiluminescence polymerase chain reaction detection of genetically modified organisms

    International Nuclear Information System (INIS)

    With the development of biotechnology, more and more genetically modified organisms (GMOs) have entered commercial market. Because of the safety concerns, detection and characterization of GMOs have attracted much attention recently. Electrochemiluminescence (ECL) method is a chemiluminescent (CL) reaction of species generated electrochemically on an electrode surface. It is a highly efficient and accurate detection method. In this paper, ECL polymerase chain reaction (PCR) combined with two types of nucleic acid probes hybridization was applied to detect GMOs for the first time. Whether the organisms contain GM components was discriminated by detecting the cauliflower mosaic virus 35S (CaMV35S) promoter and nopaline synthase (NOS) terminator. The experiment results show that the detection limit is 100 fmol of PCR products. The promoter and the terminator can be clearly detected in GMOs. The method may provide a new means for the detection of GMOs due to its simplicity and high efficiency

  4. Electrochemiluminescence polymerase chain reaction detection of genetically modified organisms

    Energy Technology Data Exchange (ETDEWEB)

    Liu Jinfeng [Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China); Xing Da [Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China)]. E-mail: xingda@scnu.edu.cn; Shen Xingyan [Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China); Zhu Debin [Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China)

    2005-04-29

    With the development of biotechnology, more and more genetically modified organisms (GMOs) have entered commercial market. Because of the safety concerns, detection and characterization of GMOs have attracted much attention recently. Electrochemiluminescence (ECL) method is a chemiluminescent (CL) reaction of species generated electrochemically on an electrode surface. It is a highly efficient and accurate detection method. In this paper, ECL polymerase chain reaction (PCR) combined with two types of nucleic acid probes hybridization was applied to detect GMOs for the first time. Whether the organisms contain GM components was discriminated by detecting the cauliflower mosaic virus 35S (CaMV35S) promoter and nopaline synthase (NOS) terminator. The experiment results show that the detection limit is 100 fmol of PCR products. The promoter and the terminator can be clearly detected in GMOs. The method may provide a new means for the detection of GMOs due to its simplicity and high efficiency.

  5. 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.

  6. Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86

    Directory of Open Access Journals (Sweden)

    Jongejan Frans

    2009-12-01

    Full Text Available Abstract Background For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility. Results The transcription levels of nine potential reference genes: β-actin (ACTB, β-tubulin (BTUB, elongation factor 1α (ELF1A, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, glutathione S-transferase (GST, H3 histone family 3A (H3F3A, cyclophilin (PPIA, ribosomal protein L4 (RPL4 and TATA box binding protein (TBP were measured in all life stages of R. microplus and R. appendiculatus. ELF1A was found to be the most stable expressed gene in both species following analysis by both geNorm and Normfinder software applications, GST showed the least stability. The expression profile of Bm86 in R. appendiculatus and R. microplus revealed a more continuous Bm86 antigen abundance in R

  7. RT-PCR方法检测犬瘟热的流行情况%Epidemic of Canine Distemper Virus was Detected by RT-PCR

    Institute of Scientific and Technical Information of China (English)

    裘云云; 施朋飞; 夏飞琴; 张保新; 赵凡凡; 王晓杜

    2012-01-01

    犬瘟热是由犬瘟热病毒引起的犬科烈性传染病,给患病动物带来重大危害.本研究基于犬瘟热病毒的NP蛋白基因设计特异性引物,利用RT-PCR获得287 bp目的片段,经TA克隆并测序.结果表明,该片段与NCBI上公布的犬瘟热病毒NP序列(登录号:EU716322)同源性达到98%.利用该特异性的RT-PCR方法,检测杭州市及周边部分地区犬瘟热的流行情况,结果表明,整个杭州及周边地区犬瘟热病毒总阳性检出率为6.7%,杭州城区阳性个体数最多,阳性率最高达到18.8%,其他地方(临安、台州、舟山)阳性率较低.该研究为犬瘟热的综合防制提供基础数据.%Canine distemper caused by canine distemper virus (CDV) is acute infection disease of canine, which caused severe losses. In this study, specific primers recognized CDV nucleoprotein were designed, the 287 bp production was amplified by RT-PCR. This production was cloned by TA cloning and sequenced, the results showed that there was a high homogeneity of 98% in nucleotide with canine distemper virus NP sequence (accession number; EU716322) published in the GenBank. Epidemic of canine distemper in Hangzhou and other cities nearby was investigated by this specific RT-PCR. The results showed that total positive detection rate of CDV was 6.7%, the number of positive individuals in Hangzhou city was largest and positive detection rate was most up to 18. 8% o positive detection of other cities (Linan, Taizhou, Zhoushan) were lower. The study is basic to prevention and treatment of canine distemper.

  8. Human coronavirus ocurrence in different populations of Sao Paulo: a comprehensive nine-year study using a pancoronavirus RT-PCR assay

    Directory of Open Access Journals (Sweden)

    Tatiane K. Cabeça

    2013-01-01

    Full Text Available Human coronaviruses (HCoVs are considered one of the most common respiratory viruses associated with respiratory tract illnesses. An emergent human coronavirus was identified as the causal agent of an epidemic of severe acute respiratory syndrome (SARS during 2002-2003. The severity of the disease combined with its rapid spread requires the continuous surveillance of coronaviruses in worldwide populations. Epidemiological and clinical data of HCoVs infectious in the Brazilian population are scarce and restricted to one or two groups of patients. Our study aimed to investigate retrospectively the presence of HCoVs in different populations of São Paulo presenting acute respiratory tract infections (ARIs during the years of 2001-2010. A pancoronavirus RT-PCR was performed in this study. Coronaviruses were detected in 126 (11.5% of 1,087 specimens. Peaks detection frequency was observed during 2002-2004 and 2008-2009, with the highest detection in 2008. The prevalence of HCoVs was higher among children with heart diseases (24.6%, patients under stem cell transplantation program (24.3% and renal transplanted patients (20.2%. Coryza, cough and fever were the most common symptoms at presentation of positive cases and wheezing, a lower respiratory tract infection symptom was reported by 12% of the total, and 27% of high at-risk patients. HCoVs may have an important role among patients with underlying conditions and transplanted ones.

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

    Science.gov (United States)

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

    2013-01-01

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

  10. RT-PCR Method for Determination of Multidrug Resistance Associated Protein(MRP) Gene in Acute Leukemia%RT-PCR检测急性白血病MRP基因

    Institute of Scientific and Technical Information of China (English)

    赵新东; 孙维波; 吴少玲

    2003-01-01

    目的探讨急性白血病(AL)患者多药耐药相关蛋白(MRP)基因表达与临床耐药的关系.方法应用半定量逆转录聚合酶链反应(RT-PCR),检测了48例AL患者骨髓和10例正常对照骨髓细胞MRP基因的表达.将MRP/β2-MG≥0.3表示为MRP阳性表达.结果复发难治组MRP的平均表达水平及阳性率最高,与正常对照组、初治组的MRP平均表达水平及阳性率相比均有显著差异(P<0.05),初治组MRP阳性病例与MRP阴性病例的首次完全缓解率(分别为28.6%和71%)之间有显著差异.结论MRP基因过度表达可导致临床耐药,是AL患者预后的重要不利因素.

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Development of a multiplex RT-PCR assay for the identification of recombination types at different genomic regions of vaccine-derived polioviruses.

    Science.gov (United States)

    Dimitriou, T G; Kyriakopoulou, Z; Tsakogiannis, D; Fikatas, A; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

    2016-08-01

    Polioviruses (PVs) are the causal agents of acute paralytic poliomyelitis. Since the 1960s, poliomyelitis has been effectively controlled by the use of two vaccines containing all three serotypes of PVs, the inactivated poliovirus vaccine and the live attenuated oral poliovirus vaccine (OPV). Despite the success of OPV in polio eradication programme, a significant disadvantage was revealed: the emergence of vaccine-associated paralytic poliomyelitis (VAPP). VAPP is the result of accumulated mutations and putative recombination events located at the genome of attenuated vaccine Sabin strains. In the present study, ten Sabin isolates derived from OPV vaccinees and environmental samples were studied in order to identify recombination types located from VP1 to 3D genomic regions of virus genome. The experimental procedure that was followed was virus RNA extraction, reverse transcription to convert the virus genome into cDNA, PCR and multiplex-PCR using specific designed primers able to localize and identify each recombination following agarose gel electrophoresis. This multiplex RT-PCR assay allows for the immediate detection and identification of multiple recombination types located at the viral genome of OPV derivatives. After the eradication of wild PVs, the remaining sources of poliovirus infection worldwide would be the OPV derivatives. As a consequence, the immediate detection and molecular characterization of recombinant derivatives are important to avoid epidemics due to the circulation of neurovirulent viral strains. PMID:27098645

  13. 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.

  14. Gene Cloning of PME from Citrus Peels by RT-PCR%RT-PCR法克隆柑橘皮果胶酯酶基因

    Institute of Scientific and Technical Information of China (English)

    叶华; 马力

    2011-01-01

    试验利用RT-PCR技术将新鲜柑橘皮果胶酯酶基因克隆到pGEM T-Easy载体上,并进行了鉴定和序列测定.结果表明:该基因为克隆成功的果胶酯酶基因,全长1797bp,开放阅读框架长1785bp,共编码594个氨基酸.该序列含有PMEI和Pectinseterase两个保守序列.该序列与Coyadonga R发表的citrus CsPME4 3'端序列氨基酸同源性100%.%The PME gene was amplified from citrus peels by RT-PCR technique and inserted into plasmid Pgem T-Easy. The interest fragment was identified and sequenced. The results showed that it presented 100% identity with the 3'extremitycitrus of CsPME4. It has 1797bp and its ORF is 1785bp which coding 594 amino acid. It has two conserved sequences: PMEI and Pectinseterase. These suggest that is PME gene from citrus peels.

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

    Science.gov (United States)

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

    2008-02-01

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

  16. Detección de biotipos del Virus de la Diarrea Viral Bovina (VDVB a través de RT-PCR

    Directory of Open Access Journals (Sweden)

    Hernán Burbano C.

    2006-06-01

    Full Text Available En el presente artículo se describe la normalización de la prueba de RT-PCR para ser empleada como herramienta en la detección del Virus de la Diarrea Viral Bovina (VDVB. Los iniciadores utilizados amplificaron un sector de 280 pb que se encuentra dentro del extremo 5’ UTR del genoma viral. Para el proceso de normalización, se usó como control positivo, cepas de referencia de VDVB (NADL, Osloss. Para evaluar reacción cruzada se usó una cepa de virus de Enfermedad de las Fronteras (BD. La obtención del cDNA se realizó por el método de «random primers» (iniciadores aleatorios. La prueba detectó tanto cepas citopáticas, como no citopáticas del VDVB. El protocolo establecido demostró un buen funcionamiento in vitro y es la base para posteriores pruebas de validación y evaluación de la prueba diagnóstica.

  17. Identification of reference genes suitable for qRT-PCR in grapevine and application for the study of the expression of genes involved in pterostilbene synthesis.

    Science.gov (United States)

    Gamm, Magdalena; Héloir, Marie-Claire; Kelloniemi, Jani; Poinssot, Benoît; Wendehenne, David; Adrian, Marielle

    2011-04-01

    The recent publication of the grapevine genome sequence facilitates the use of qRT-PCR to study gene expression changes. For this approach, reference genes are commonly used to normalize data and their stability of expression should be systematically validated. Among grapevine defenses is the production of the antimicrobial stilbenic phytoalexins, notably the highly fungitoxic pterostilbene, which plays a crucial role in grapevine interaction with Plasmopara viticola and Botrytis cinerea. As a resveratrol O-methyltransferase (ROMT) gene involved in pterostilbene synthesis was recently identified, we investigated the accumulation of the corresponding transcripts to those of two other stilbene biosynthesis related genes phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) in response to pathogen infection. Using three computer-based statistical methods and C(t) values or LRE method generated values as input data, we have first identified two reference genes (VATP16 and 60SRP) suitable for normalization of qPCR expression data obtained in grapevine leaves and berries infected by P. viticola and B. cinerea, respectively. Next, we have highlighted that the expression of ROMT is induced in P. viticola-infected leaves and also in B. cinerea-infected berries, confirming the involvement of pterostilbene in grapevine defenses. PMID:21340517

  18. Development and preliminary evaluation of a real-time polymerase chain reaction for the identification of Culicoides obsoletus sensu strictu, C. scoticus and C. montanus in the Obsoletus Complex in Italy

    Directory of Open Access Journals (Sweden)

    Federica Monaco

    2010-06-01

    Full Text Available A real-time polymerase chain reaction (PCR using a green fluorescence dye, followed by a comparison of derivative melting curves in the post-amplification phase, was developed to distinguish species of Culicoides within the Obsoletus Complex. The selected target sequence was internal transcribed spacer 2 (ITS 2 of the ribosomal DNA (rDNA. Using the newly developed method, 140 midges were morphologically classified in the Obsoletus Complex and were processed. The results were compared to those obtained by combining the morphological identification with the gel based reverse transcriptase (RT-PCR. By analysing the species-specific pattern of the dissociation curves, it was possible to identify 52 midges as Culicoides scoticus, 82 midges as C. obsoletus sensu strictu and 6 as C. montanus. These results matched those obtained by the combination of gel-based PCR and morphological identification used on a routine basis. Given its diagnostic flexibility, rapid results, automation capability, high quality result performance and expression, the real-time ITS 2 rDNA PCR appears to be more functional and efficient than the gel-based PCR, especially when dealing with large-scale monitoring of midges belonging to the Obsoletus Complex.

  19. Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples

    Directory of Open Access Journals (Sweden)

    Negi S

    2007-01-01

    Full Text Available Purpose: The correlation between the presence of specific gene sequence of M. tuberculosis and specific diagnosis of clinical tuberculosis is not known. This study compared the results of polymerase chain reaction (PCR amplification of M . tuberculosis specific DNA sequences (IS6110, 65kDa, 38kDa and mRNA coding for 85 B protein from different clinical samples of pulmonary and extrapulmonary tuberculosis. Methods: One hundred and seventy-two clinical samples from suspected tuberculosis patients were tested for smear examination, culture (LJ and rapid BACTEC 460 TB system and PCR. PCR was performed with specific primers for the targets: IS6110, 65kDa, 38kDa and 85B. Results: Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture ( P < 0.05. Smear positive samples (56 and the samples (36 showing positive results by conventional methods (smear and LJ medium culture and BACTEC were found to be positive by all PCR protocols. No significant difference was found between the four PCR protocols ( P >0.05. The primer specific for amplifying the 123bp IS6110 fragment gave the highest positivity (83%, followed by 65kDa, 38kDa and 85B RT-PCR in descending order. Conclusions: These data suggest that the presence of IS6110 correlates more closely with the diagnosis of clinical tuberculosis than that of 65kDa, 38kDa and 85B proteins.

  20. A RT-PCR method for selective amplification and phenotypic characterization of all three serotypes of Sabin-related polioviruses from viral mixtures

    Directory of Open Access Journals (Sweden)

    Eliane Veiga da Costa

    2012-08-01

    Full Text Available Outbreaks caused by vaccine-derived polioviruses are challenging the final eradication of paralytic poliomyelitis. Therefore, the surveillance of the acute flaccid paralysis cases based on poliovirus isolation and characterization remains an essential activity. Due to the use of trivalent oral poliovirus vaccine (OPV, mixtures containing more than one serotype of Sabin-related polioviruses are frequently isolated from clinical samples. Because each poliovirus isolate needs to be individually analyzed, we designed polymerase chain reaction primers that can selectively distinguish and amplify a genomic segment of the three Sabin-related poliovirus serotypes present in mixtures, thus, optimizing the diagnosis and providing prompt information to support epidemiologic actions.

  1. 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.

  2. Polymerase chain reaction: A molecular diagnostic tool in periodontology

    Science.gov (United States)

    Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

    2016-01-01

    This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease. PMID:27143822

  3. Early detection of typhoid by polymerase chain reaction

    International Nuclear Information System (INIS)

    Typhoid is a common problem in developing countries. Cultivation ofbacteria and serology (especially Widal test) gives unacceptable levels offalse-negative and false-positive results respectively. In this study, arecently introduced polymerase chain reaction based technique (which has 100%specificity for Salmonella typhi) was compared with blood culture and Widaltest during the first week of illness of 82 suspected cases of typhoid. Therespective figures of positivity for PCR, blood culture and Widal test were71.95%, 34.1% and 36.5%. A control group of 20 healthy persons gave figuresof 0%, 0% and 33.3%, respectively. We conclude that this PCR-based techniqueis not only absolutely specific, but also very sensitive and therefore muchsuperior to blood culture and, Widal test for the early diagnosis of typhoid.(author)

  4. Polymerase chain reaction-based gene removal from plasmids

    Directory of Open Access Journals (Sweden)

    Vishnu Vardhan Krishnamurthy

    2015-09-01

    Full Text Available This data article contains supplementary figures and methods to the research article entitled, “Multiplex gene removal by two-step polymerase chain reactions” (Krishnamurthy et al., Anal. Biochem., 2015, doi:http://dx.doi.org/10.1016/j.ab.2015.03.033, which presents a restriction-enzyme free method to remove multiple DNA segments from plasmids. Restriction-free cloning methods have dramatically improved the flexibility and speed of genetic manipulation compared to conventional assays based on restriction enzyme digestion (Lale and Valla, 2014. DNA Cloning and Assembly Methods, vol. 1116. Here, we show the basic scheme and characterize the success rate for single and multiplex gene removal from plasmids. In addition, we optimize experimental conditions, including the amount of template, multiple primers mixing, and buffers for DpnI treatment, used in the one-pot reaction for multiplex gene removal.

  5. Polymerase chain reaction of Au nanoparticle-bound primers

    Institute of Scientific and Technical Information of China (English)

    SHEN Hebai; HU Min; YANG Zhongnan; WANG Chen; ZHU Longzhang

    2005-01-01

    Polymerase chain reaction (PCR) is a useful technique for in vitro amplification of a DNA fragment. In this paper, a PCR procedure using Au nanoparticle (AuNP) -bound primers was systemically studied. The 5′-SH- (CH2)6-modified primers were covalently attached to the AuNP surface via Au-S bonds, and plasmid pBluescript SK was used as a template. The effects of the concentration of AuNP-bound primers, annealing temperature and PCR cycles were evaluated, respectively. The results indicate that PCR can proceed successfully under optimized condition, with either forward or reverse primers bound to the AuNP surface or with both the two primers bound to the AuNP surface. Development of PCR procedure based on AuNPs not only makes the isolation of PCR products very convenient, but also provides novel methods to prepare AuNP-bound ssDNA and nanostructured material.

  6. Enhancing the efficiency of polymerase chain reaction using graphene nanoflakes

    International Nuclear Information System (INIS)

    The effect of the recently developed graphene nanoflakes (GNFs) on the polymerase chain reaction (PCR) has been investigated in this paper. The rationale behind the use of GNFs is their unique physical and thermal properties. Experiments show that GNFs can enhance the thermal conductivity of base fluids and results also revealed that GNFs are a potential enhancer of PCR efficiency; moreover, the PCR enhancements are strongly dependent on GNF concentration. It was found that GNFs yield DNA product equivalent to positive control with up to 65% reduction in the PCR cycles. It was also observed that the PCR yield is dependent on the GNF size, wherein the surface area increases and augments thermal conductivity. Computational fluid dynamics (CFD) simulations were performed to analyze the heat transfer through the PCR tube model in the presence and absence of GNFs. The results suggest that the superior thermal conductivity effect of GNFs may be the main cause of the PCR enhancement. (paper)

  7. Nested methylation-specific polymerase chain reaction cancer detection method

    Science.gov (United States)

    Belinsky, Steven A.; Palmisano, William A.

    2007-05-08

    A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

  8. Modelling of Serpentine Continuous Flow Polymerase Chain Reaction Microfluidics

    Directory of Open Access Journals (Sweden)

    Abubakar Mohammed

    2012-03-01

    Full Text Available The continuous flow Polymerase Chain Reaction (PCR microfluidics DNA amplification device is a recent discovery aimed at eliminating the cyclic hold experienced while using the alternative stationary device.The Application of Computational Fluid Dynamics is increasingly growing and can help achieve optimal designs before actual fabrication. This paper presents a CFD modelling of a continuous flow serpentine PCR device with narrow and wider channels. There are two temperature regions at 950C and 600C for denaturation and annealing respectively. Extension is achieved along the middle of the channel at 720C owing to temperature gradient. The model require a pressure of 42.6KPa for a 30 cycle amplification.

  9. Polymerase chain reaction: A molecular diagnostic tool in periodontology.

    Science.gov (United States)

    Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

    2016-01-01

    This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease. PMID:27143822

  10. Polymerase chain reaction: A molecular diagnostic tool in periodontology

    Directory of Open Access Journals (Sweden)

    Rajendran Maheaswari

    2016-01-01

    Full Text Available This review discusses the principles of polymerase chain reaction (PCR and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease.

  11. Establishment and Initial Application of RT- PCR for Quick Detectionof Bovine Viral Diarrhea Virus%牛病毒性腹泻病毒反转录PCR快速检测方法的建立与初步应用

    Institute of Scientific and Technical Information of China (English)

    李倬; 田镔; 李明生; 平玲

    2012-01-01

    根据GenBank中登录的牛病毒性腹泻病毒(BVDV)5'非编码区基因序列,设计合成了1对特异性引物,建立了检测BVDV的反转录PCR快速检测方法.通过对该方法的特异性、敏感性和重复性进行试验,结果显示,该方法从BVDV标准毒株Oregon C24V中扩增出267 bp的特异性片段,该方法重复性好,反应批内检测结果相同.与牛轮状病毒、牛冠状病毒、猪瘟病毒和F4新生牛肾传代正常细胞无交叉反应,具有高度的特异性,而且敏感性高,最低检出限为10~1.84 TCID50/mL.利用该方法对42份临床腹泻病牛疑似粪便样品进行了检测,结果检出7份阳性,而同时利用IDEXX公司抗原检测试剂盒检出阳性只有6份.表明,建立的该方法具有快速、敏感、特异等优点,是牛病毒性腹泻病毒病的临床诊断和流行病学调查的有力工具.%A reverse transcription- coupled PCR(RT- PCR) assay for quick detection of bovine viral diarrhea virus(BVDV) was established using a pair of specific primers based on the 5"UTR gene of BVDV published in GenBank. The results showed that this method could specifically amplify a 267 bp fragment from BVDV Oregon C24 V strain, reproducibility of this assay were reliable and the results were fully consistent. The specificity test proved that this assay had a high specificity which had no cross- reaction with bovine rotavirus, bovine coronavirus, classical swine fever virus and normal F4 NBPC cells. The assay also had good sensitivity, and the detection limit was up to 10-- 1.84 TCID50/mL. 7 positive of 42 samples from clinical diarrhea bovine were detected by RT- PCR and only 6 positive were detected by IDEXX diagnostic kit test at the same time. The results revealed that established RT - PCR assay possessed some advantages such as fast, sensitive and specific. It may be used for clinical diagnosis and the epidemiologic survey of bovine viral diarrhea/mucosal disease as a powerful tool.

  12. Perfil de restrição de um fragmento do gene da hemaglutinina amplificado pela RT-PCR a partir de estirpes vacinais e selvagens do vírus da cinomose canina Restriction pattern of a hemagglutinin gene amplified by RT-PCR from vaccine strains and wild-type canine distemper virus

    Directory of Open Access Journals (Sweden)

    F.J. Negrão

    2006-12-01

    Full Text Available Fragmentos com 721 pares de bases do gene da hemaglutinina (H do vírus da cinomose canina (CDV, amplificados pela RT-PCR a partir de três estirpes vacinais (Snyder Hill, Onderstepoort e Rockborn e de 27 amostras de campo, provenientes de cães com cinomose, foram clivados com as endonucleases Hinf I and Rsa I. A seleção das enzimas foi realizada por meio de análises in silico de seqüências do CDV depositadas em bases públicas de dados. Tanto as estirpes vacinais quanto as amostras selvagens do CDV apresentaram com a enzima Hinf I o mesmo perfil de restrição, confirmando a identidade do fragmento amplificado pela RT-PCR, uma vez que todas as estirpes com seqüências disponíveis (GenBank têm sítios de restrição para essa enzima nas mesmas posições. O perfil de restrição das estirpes vacinais Snyder Hill e Onderstepoort, que diferem entre si, foi confirmado com a enzima Rsa I que também clivou a estirpe Rockborn nas mesmas posições que a estirpe Snyder Hill. Todas as 27 amostras de campo do CDV apresentaram com a enzima Rsa I o mesmo perfil de restrição, indicando conservar os mesmos sítios de restrição para essa enzima. O perfil das amostras de campo foi diferente daquele obtido nas três estirpes vacinais. Os perfis de restrição do gene que codifica a hemaglutinina do CDV, gerados pela enzima Rsa I, sugerem diferenças moleculares entre as estirpes vacinais e as selvagens circulantes na região norte do estado do Paraná e abrem a perspectiva da elaboração de análises moleculares comparativas mais complexas, como o seqüenciamento de todo o gene H, de estirpes do CDV identificadas em diferentes regiões brasileiras.The restriction fragment length polymorphism (RFLP assay of a 721 bp fragment from hemagglutinin (H gene of canine distemper virus (CDV amplified by RT-PCR was analyzed with Hinf I and Rsa I enzymes. Clinical samples from 27 dogs with natural canine distemper infection, and three vaccine (Snyder Hill

  13. 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

    Full Text Available Neospora caninum é um dos principais agentes causadores de abortamentos e natimortalidade em bovinos. A defesa imune do hospedeiro é capaz de inibir a atividade dos taquizoítos na fase aguda da infecção, mas não age sobre os bradizoítos nos cistos teciduais. A ativação e a modulação dessa resposta de defesa são controladas por mediadores celulares. A técnica do RT-PCR em tempo real foi empregada para a detecção de alguns desses mediadores durante a infecção pelo N. caninum. Foram analisadas amostras de linfonodos poplíteos, fígado e córtex cerebral de bezerros Holandeses e Nelores infectados com taquizoítos por via intramuscular e controles não-infectados, abatidos no sexto dia pós-inoculação. A RT-PCR em tempo real detectou a expressão dos genes em todos os tecidos analisados. Não houve variação significativa na expressão do gene GADPH entre os grupos, a eficiência de amplificação desse foi similar aos demais genes testados e foi empregado como controle endógeno na análise. A comparação entre infectados e não-infectados permitiu a quantificação relativa da expressão gênica. A expressão dos genes IFN-γ e TNF-α apresentou elevação significante em algumas amostras. Os genes iNOS e TGF-β1 apresentaram algumas variações não-significativas e os valores de IL-4 e IL-10 permaneceram praticamente inalterados.Neospora caninum is one of the main causes of abortion and natimortality in cattle. Host immune defense is capable to inhibit tachyzoite activity during acute infection, but there is no action against bradyzoites in tissue cysts. Activation and modulation of this response is controlled by cell mediators. The real-time RT-PCR technique was employed to detect some of those mediators during N. caninum infection. Holstein and Nelore calves intramuscularly infected with tachyzoites and uninfected controls were slaughtered at the sixth day post-infection and popliteal lymph node, liver and brain cortex

  14. Effect of captopril and paraquat on expression of p53 and Bcl-2 genes and proteins in rat lung tissue using RT-PCR and immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Azizi E

    2008-10-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Paraquat is an herbicide produced and used prevalently worldwide. Studies have shown that lung fibrosis induced by paraquat can be prevented or delayed by certain antioxidants, iron chelating agents, melatonin, and, recently, blood pressure lowering drugs such as captopril."n"n Methods: The protective effects of captopril on paraquat toxicity were studied using RT-PCR and immunohistochemistry to determine the gene and protein expression of p53 and Bcl-2 in lung tissue samples from rats treated with captopril before and after exposure to paraquat."n"n Results: We found no significant difference in the gene and protein expression of p53 in different tissue samples, except for mRNA levels in the lung tissue of captopril-treated rats. However, the protein expression of Bcl-2 is greater in tissue from rats exposed to paraquat alone and paraquat together with pre- and posttreatment with captopril compared to tissue from untreated control rats and from those treated with captopril alone, which can be due to inflammatory responses of lung tissue. By RT-PCR, we were unable to detect Bcl-2 in lung tissue samples."n"n Conclusion: These results show that paraquat does not induce significant DNA damage; therefore, the

  15. First Molecular Detection of Group A Rotavirus in Urban and Hospital Sewage systems by Nested-RT PCR in Shiraz, Iran

    Directory of Open Access Journals (Sweden)

    Yahya Tahamtan

    2013-05-01

    Full Text Available Group A rotaviruses are the most significant cause of acute gastroenteritis in children worldwide. Rotaviruses are shed in high numbers and dispersed widely throughout bodies of water in the environment. This represents a significant health hazard for humans, mainly due to the stability of the viruses during wastewater treatment processes. This study was conducted to evaluate the prevalence of rotaviruses, to determine G genotypes of circulating rotaviruses and to assess the efficiency of rotavirus removal in urban and hospital sewage treatment plants in Shiraz, Iran.Materials and methods: During the period from October 2010 to June 2011, a total of sixty sewage samples from urban and hospital sewage disposal systems were collected by Grab Sampling in Shiraz, Iran. All the samples were concentrated in pellet form and two-phase methods and then group A rotaviruses were investigated with enzyme immunoassays (EIA. Rotavirus-positive specimens were genotyped by the nested RT-PCR and by using different types of specific primers.Results:In total, rotaviruses were identified in 25% (15 cases of sewage samples, representing 73.33% (11 cases of influent and 26.67% (4 cases of effluent systems. The frequency of rotavirus detection in autumn, winter and spring was 46.67%, 33.33% and 20%, respectively (P= 0.004. The most common circulating genotype was G1 (73.33%, followed by G1G4 (20% and non-typeable (6.67%, respectively.Conclusions:The high prevalence of rotaviruses in urban and hospital sewage systems highlights the importance of environmental surveillance as a tool to detect new genotypes and to investigate the epidemiology of rotaviruses circulating in the community.

  16. Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR

    International Nuclear Information System (INIS)

    Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles and oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (VvBAS) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the VvBAS increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals

  17. Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L..

    Directory of Open Access Journals (Sweden)

    Candy M Taylor

    Full Text Available Quantitative Reverse Transcription PCR (qRT-PCR is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC, Helicase (HEL, and Polypyrimidine tract-binding protein (PTB] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other

  18. Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

    Science.gov (United States)

    Taylor, Candy M; Jost, Ricarda; Erskine, William; Nelson, Matthew N

    2016-01-01

    Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more

  19. Detecting mycoplasma contamination in cell cultures by polymerase chain reaction.

    Science.gov (United States)

    Uphoff, Cord C; Drexler, Hans G

    2011-01-01

    The detection of mycoplasmas in human and animal cell cultures is mandatory for every cell culture laboratory, because these bacteria are common contaminants, persist unrecognized in cell cultures for many years, and affect research results as well as the purity of cell culture products. The reliability of the mycoplasma detection depends on the sensitivity and specificity of the method and should also be convenient to be included in the basic routine of cell culture quality assessment. Polymerase chain reaction (PCR) detection is one of the acknowledged methodologies to detect mycoplasmas in cell cultures and cell culture products. Although the PCR offers a fast and simple technique to detect mycoplasmas, the method is also susceptible to errors and can produce false positive as well as false-negative results. Thus, the establishment and the routine application of the PCR assay require optimization and the inclusion of the appropriate control reactions. The presented protocol describes sample preparation, DNA extraction, PCR run, the analysis of the PCR products, and speciation of the contaminant. It also provides detailed information on how to avoid artifacts produced by the method. Established properly, PCR is a reliable, fast, and sensitive method and should be applied regularly to monitor the contamination status of cell cultures. PMID:21516400

  20. Controlling Hybridization Chain Reactions with pH.

    Science.gov (United States)

    Idili, Andrea; Porchetta, Alessandro; Amodio, Alessia; Vallée-Bélisle, Alexis; Ricci, Francesco

    2015-08-12

    By taking inspiration from nature, where self-organization of biomolecular species into complex systems is finely controlled through different stimuli, we propose here a rational approach by which the assembly and disassembly of DNA-based concatemers can be controlled through pH changes. To do so we used the hybridization chain reaction (HCR), a process that, upon the addition of an initiator strand, allows to create DNA-based concatemers in a controlled fashion. We re-engineered the functional units of HCR through the addition of pH-dependent clamp-like triplex-forming domains that can either inhibit or activate the polymerization reaction at different pHs. This allows to finely regulate the HCR-induced assembly and disassembly of DNA concatemers at either basic or acidic pHs in a reversible way. The strategies we present here appear particularly promising as novel tools to achieve better spatiotemporal control of self-assembly processes of DNA-based nanostructures. PMID:26177980

  1. Circulating polymerase chain reaction chips utilizing multiple-membrane activation

    Science.gov (United States)

    Wang, Chih-Hao; Chen, Yi-Yu; Liao, Chia-Sheng; Hsieh, Tsung-Min; Luo, Ching-Hsing; Wu, Jiunn-Jong; Lee, Huei-Huang; Lee, Gwo-Bin

    2007-02-01

    This paper reports a new micromachined, circulating, polymerase chain reaction (PCR) chip for nucleic acid amplification. The PCR chip is comprised of a microthermal control module and a polydimethylsiloxane (PDMS)-based microfluidic control module. The microthermal control modules are formed with three individual heating and temperature-sensing sections, each modulating a specific set temperature for denaturation, annealing and extension processes, respectively. Micro-pneumatic valves and multiple-membrane activations are used to form the microfluidic control module to transport sample fluids through three reaction regions. Compared with other PCR chips, the new chip is more compact in size, requires less time for heating and cooling processes, and has the capability to randomly adjust time ratios and cycle numbers depending on the PCR process. Experimental results showed that detection genes for two pathogens, Streptococcus pyogenes (S. pyogenes, 777 bps) and Streptococcus pneumoniae (S. pneumoniae, 273 bps), can be successfully amplified using the new circulating PCR chip. The minimum number of thermal cycles to amplify the DNA-based S. pyogenes for slab gel electrophoresis is 20 cycles with an initial concentration of 42.5 pg µl-1. Experimental data also revealed that a high reproducibility up to 98% could be achieved if the initial template concentration of the S. pyogenes was higher than 4 pg µl-1. The preliminary results of the current paper were presented at the 19th IEEE International Conference on Micro Electro Mechanical Systems (IEEE MEMS 2006), Istanbul, Turkey, 22-26 January, 2006.

  2. Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S M; Danganan, L; Tammero, L; Vitalis, B; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed candidate multiplexed assays that may potentially be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the ability to improve our nation's capability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect food and agricultural resources with a diagnostic test which could enhance the nation's capabilities for early detection of a foreign animal disease. In FY2005 with funding from the DHS, LLNL developed the first version (Version 1.0) of a multiplexed (MUX) nucleic-acid-based RT-PCR assay that included signatures for foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases (FADs) of swine, Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease Virus (SVDV), and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus [BPSV], Orf of sheep, and Pseudocowpox). In FY06, LLNL has developed Bovine and Porcine species-specific panel which included existing signatures from Version 1.0 panel as well as new signatures. The MUX RT-PCR porcine assay for detection of FMDV includes the FADs, VESV and SVD in addition to vesicular stomatitis virus (VSV) and porcine reproductive and respiratory syndrome (PRRS). LLNL has also developed a MUX RT-PCR bovine assay for detection of FMDV with rule out tests for the two bovine FADs malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis

  3. A REAL-TIME RT-PCR REACTION SPECIFIC FOR BOVINE ENTEROVIRUSES PROVIDES A RAPID ASSAY FOR FECAL CONTAMINATION OF THE ENVIRONMENT BY CATTLE

    Science.gov (United States)

    Characterization of bovine enteroviruses found in a closed herd of cattle and the surrounding environment indicated that bovine enteroviruses (BEV) could serve as useful markers of contamination with cattle waste. BEV was found in feces from 76% of cattle, 38% of white-tail deer in the same area an...

  4. Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

    Directory of Open Access Journals (Sweden)

    Seiger Keirsten

    2008-10-01

    Full Text Available Abstract Background HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. Methods Standard operating procedures for each assay were established. HLA-A2 (A*0201 tetramer assay specific for gp100209(210M and MART-126–35(27L, IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520 or MART-1 (TIL 1143 and TIL1235, were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127–35, or influenza-M1 and negative control peptide HIV to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD and limit of quantification (LOQ. In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. Results The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV Conclusion Our results demonstrated the tetramer flow cytometry assay, IFN

  5. Analysis of hepcidin expression: In situ hybridization and quantitative polymerase chain reaction from paraffin sections

    OpenAIRE

    2012-01-01

    AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC).

  6. Effects of upconversion nanoparticles on polymerase chain reaction.

    Directory of Open Access Journals (Sweden)

    Sang-Hyun Hwang

    Full Text Available Nanoparticles (NPs are attractive materials owing to their physical and electrochemical properties, which make them extremely useful in diagnostic applications. Photon upconversion is the phenomenon where high-energy photons are emitted upon excitation of low-energy photons. Nucleic acids detection based on upconversion nanoparticles (UCNPs, which display a high signal-to-noise ratio and no photobleaching, has been widely applied. We evaluated whether UCNPs can improve polymerase chain reaction (PCR specificity and affect PCR amplification. The effects of UCNPs with a diameter size of 40, 70, and 250 nm were evaluated using 3 PCR kits (AccuPower PCR PreMix, AmpliTaq Gold 360 Master Mix, and HotStarTaq Plus Master Mix and 3 real-time PCR kits (AccuPower GreenStar qPCR PreMix, SYBR Green PCR Master Mix, and QuantiTect SYBR Green PCR Kit. Quantum dots were used for comparison with the UCNPs. In the presence of an appropriate concentration of UCNPs, PCR specificity was optimized. UCNPs of 40-nm size improved PCR specificity more effectively than did UCNPs sized 70 or 250 nm. As the size and concentrations of the UCNPs were increased, PCR amplification was more severely inhibited. At lower annealing temperatures (25°C-45°C, addition of the 40 nm UCNP (1 µg/µL to the PCR reagent produced specific PCR products without nonspecific sequence amplification. Therefore, UCNPs of different sizes, with different DNA polymerases used in the commercial kits, showed different inhibitory effects on PCR amplification. These results demonstrate that optimization of UCNPs, added to reaction mixtures at appropriate concentrations, can improve PCR specificity. However, the mechanism underlining UCNPs effect on PCR remains unclear and will require further investigation.

  7. Identifying of meat species using polymerase chain reaction (PCR)

    Science.gov (United States)

    Foong, Chow Ming; Sani, Norrakiah Abdullah

    2013-11-01

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one's diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  8. Identifying of meat species using polymerase chain reaction (PCR)

    International Nuclear Information System (INIS)

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one’s diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing

  9. Effects of Superparamagnetic Nanoparticle Clusters on the Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Toshiaki Higashi

    2012-04-01

    Full Text Available The polymerase chain reaction (PCR method is widely used for the reproduction and amplification of specific DNA segments, and a novel PCR method using nanomaterials such as gold nanoparticles has recently been reported. This paper reports on the effects of superparamagnetic nanoparticles on PCR amplification without an external magnetic field, and clarifies the mechanism behind the effects of superparamagnetic particle clusters on PCR efficiency by estimating the structures of such clusters in PCR. It was found that superparamagnetic nanoparticles tend to inhibit PCR amplification depending on the structure of the magnetic nanoparticle clusters. The paper also clarifies that Taq polymerase is captured in the spaces formed among magnetic nanoparticle clusters, and that it is captured more efficiently as a result of their motion from heat treatment in PCR thermal cycles. Consequently, Taq polymerase that should be used in PCR is reduced in the PCR solution. These outcomes will be applied to novel PCR techniques using magnetic particles in an external magnetic field.

  10. Identifying of meat species using polymerase chain reaction (PCR)

    Energy Technology Data Exchange (ETDEWEB)

    Foong, Chow Ming; Sani, Norrakiah Abdullah [School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor (Malaysia)

    2013-11-27

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one’s diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  11. 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.

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

    Science.gov (United States)

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

    2006-04-15

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

  13. 诺沃克病毒实时荧光RT-PCR检测方法的建立%Development of a Real-time RT-PCR for Detection of Norwalk Virus

    Institute of Scientific and Technical Information of China (English)

    邱杨; 赵丽; 卢小雨; 陈进会; 兰文生; 刘建丽; 王伟

    2012-01-01

      In this paper,a real-time RT-PCR was developed for detection of Norwalk virus,with the primers and fluorescence probe designed according to the reported genome sequence of Norwalk virus. By the Real-time RT-PCR detection,a specific fluorescence curve was got. It was shown that the test results were exacted and reliable.%  本文建立了一种用于诺沃克病毒实时荧光RT-PCR检测的引物和方法。分析已报道的诺沃克病毒基因组序列,分别设计引物和荧光探针。通过实时荧光RT-PCR检测,得到诺沃克的特异性荧光曲线,检测结果准确、可靠。

  14. Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus Hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes

    OpenAIRE

    Nerland Audun; Øvergård Aina-Cathrine; Patel Sonal

    2010-01-01

    Abstract Background Real time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, β-actin1 (ACTB...

  15. Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus Hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes

    OpenAIRE

    Øvergård, Aina-Cathrine; Nerland, Audun H.; Patel, Sonal

    2010-01-01

    Background Real time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, β-actin1 (ACTB1), e...

  16. Prevalence study of Bovine viral diarrhea virus by evaluation of antigen capture ELISA and RT-PCR assay in Bovine, Ovine, Caprine, Buffalo and Camel aborted fetuses in Iran

    OpenAIRE

    Safarpoor Dehkordi, Farhad

    2011-01-01

    Bovine viral diarrhea virus is a pestivirus in the family Flaviviridae that cause abortions and stillbirths in livestock and its traditional diagnosis is based on cell culture and virus neutralization test. In this study, for more sensitive, specific detection and determined the prevalence of virus in aborted Bovine, Ovine, Caprine, Buffalo and Camel fetuses the antigen capture ELISA and RT-PCR were recommended. From the total of 2173 aborted fetuses, 347 (15.96%) and 402 (18.49%) were positi...

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

    Science.gov (United States)

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

    2008-01-01

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

  18. Buoyancy-Driven Polymerase Chain Reaction (PCR) Devices

    Energy Technology Data Exchange (ETDEWEB)

    Ness, K D; Wheeler, E K; Benett, W; Stratton, P; Christian, A; Chen, A; Ortega, J; Weisgraber, T H; Goodson, K E

    2004-09-28

    Polymerase chain reaction (PCR) facilitates DNA detection by significantly increasing the concentration of specific DNA segments. A new class of PCR instruments uses a buoyancy-driven re-circulating flow to thermally cycle the DNA sample and benefits from reduced cycle times, low sample volumes, a miniaturized format, and low power consumption. This paper analyzes a specific buoyancy PCR device in a micro-channel ''race-track'' geometry to determine key parameters about PCR cycle times and other figures of merit as functions of device dimensions. The 1-D model balances the buoyancy driving force with frictional losses. A hydrostatic pressure imbalance concept is used between the left and right sides of the fluid loop to calculate the buoyancy driving force. Velocity and temperature distributions within the channels are determined from two-dimensional analysis of the channel section, with developing region effects included empirically through scaled values of the local Nusselt number. Good agreement between four independent verification steps validate the 1-D simulation approach: (1) analytical expressions for the thermal entrance length are compared against, (2) comparison with a full 3-D finite element simulation, (3) comparison with an experimental flow field characterization, and (4) calculation of the minimum PCR runtime required to get a positive PCR signal from the buoyancy-driven PCR device. The 1-D approach closely models an actual buoyancy-driven PCR device and can further be used as a rapid design tool to simulate buoyancy PCR flows and perform detailed design optimizations studies.

  19. The ligase chain reaction as a primary screening tool for the detection of culture positive tuberculosis.

    LENUS (Irish Health Repository)

    O'Connor, T M

    2012-02-03

    BACKGROUND: The ligase chain reaction Mycobacterium tuberculosis assay uses ligase chain reaction technology to detect tuberculous DNA sequences in clinical specimens. A study was undertaken to determine its sensitivity and specificity as a primary screening tool for the detection of culture positive tuberculosis. METHODS: The study was conducted on 2420 clinical specimens (sputum, bronchoalveolar lavage fluid, pleural fluid, urine) submitted for primary screening for Mycobacterium tuberculosis to a regional medical microbiology laboratory. Specimens were tested in parallel with smear, ligase chain reaction, and culture. RESULTS: Thirty nine patients had specimens testing positive by the ligase chain reaction assay. Thirty two patients had newly diagnosed tuberculosis, one had a tuberculosis relapse, three had tuberculosis (on antituberculous therapy when tested), and three had healed tuberculosis. In the newly diagnosed group specimens were smear positive in 21 cases (66%), ligase chain reaction positive in 30 cases (94%), and culture positive in 32 cases (100%). Using a positive culture to diagnose active tuberculosis, the ligase chain reaction assay had a sensitivity of 93.9%, a specificity of 99.8%, a positive predictive value of 83.8%, and a negative predictive value of 99.9%. CONCLUSIONS: This study is the largest clinical trial to date to report the efficacy of the ligase chain reaction as a primary screening tool to detect Mycobacterium tuberculosis infection. The authors conclude that ligase chain reaction is a useful primary screening test for tuberculosis, offering speed and discrimination in the early stages of diagnosis and complementing traditional smear and culture techniques.

  20. Comparación de dos kits de RT-PCR en la detección de ARNm de dos genes endógenos de papa (Solanum tuberosum spp. Andígena

    Directory of Open Access Journals (Sweden)

    Milet Zabaleta Vanegas

    2009-01-01

    Full Text Available RT-PCR es una técnica en la que usando ARN mensajero como molde, se obtiene complementario o cADN por transcripción inversa, y luego se amplifica uno de los cADN por PCR, mediante el uso de primers específicos. Esta técnica permite realizar estudios de expresión, a nivel de ARN mensajero. Con el propósito de implementar la técnica en papa (Solanum tuberosum spp. Andígena, se utilizaron plántulas cultivadas in vitro de la variedad Pastusa Suprema. Inicialmente se establecieron las condiciones para la extracción de ARN total usando el kit TRizol® Reagent de InvitrogenTM, con el que se obtuvieron excelentes resultados. Este ARN se usó como molde para evaluar dos kits: “ONE Step superScriptTM” y “SuperScriptâ„¢ First Strand Syntesis For RT-PCR SSâ„¢ II RT”, de InvitrogenTM. Se usaron primers específicos para dos genes endógenos: cox y actina. El primero es un gen mi­tocondrial y el segundo es un gen nuclear. Se observaron señales claras y diferenciables de amplificación para cox, utilizando el kit “ONE Step superScriptTM”, con un tamaño esperado de 96 pb. Para actina, se observó una señal clara de amplificación de 300 pb, con el kit “SuperScriptâ„¢ First Strand Syntesis For RT-PCR SSâ„¢ II RT”.  Palabras clave: Solanum tuberosum; cox, actina; RT-PCR; papa; Solanum tuberosum; cox; actin; RT-PCR; potato.

  1. 荧光RT-PCR法快速检测微量建兰花叶病毒研究%Rapid Detection of Trace Amounts of Cymbidium Mosaic Virus by Real-Time RT-PCR

    Institute of Scientific and Technical Information of China (English)

    柳爱春; 刘超; 李峰

    2011-01-01

    According to the conserved sequence of the coat protein gene of Cymbidium mosaic virus (CymMV)in the GeneBank, three pairs of primers used in real-time RT-PCR methods were designed and synthesized for detecting traces of the virus which could not be detected by ordinary RT-PCR-Agarose gel electrophoresis in spider orchid (A rachnis sp. ). Melting curve analysis showed that each amplified product was a single product form. Sequencing and homology analysis indicated that the actual sequence lengths of the amplified products were entirely consistent with expectations, and the amplified gene had between 94% to 96% homology with the corresponding gene of CymMV in GeneBank in spider orchid. Compared to ordinary normal RT-PCR,the real-time RT-PCR method characterized by high sensitivity,fast operation,intuitive and accurate,is suitable for detection of trace amounts of CymMV and early diagnosis in seedlings.%根据基因库中建兰花叶病毒外壳蛋白基因的保守序列,设计并合成3对特异性引物,利用荧光RT-PCR方法在蜘蛛兰中检测到普通RT-PCR-琼脂糖电泳法检测不到的微量病毒。熔解曲线分析表明,每一扩增产物为单一产物形态。通过测序和同源性分析,试验证实扩增产物序列的实际长度与预期完全相符。蜘蛛兰样品的扩增产物基因序列与基因库中建兰花叶病毒相应序列的同源性为94%~96%。与普通RT-PCR的相比。荧光RT-PCR方法具有灵敏度高、操作快速、结果直观准确的特点,可应用于种苗中微量建兰花叶病毒的早期检测。

  2. Evaluation of suitable reference genes for quantitative RT-PCR during development and abiotic stress in Panonychus citri (McGregor) (Acari: Tetranychidae).

    Science.gov (United States)

    Niu, Jin-Zhi; Dou, Wei; Ding, Tian-Bo; Yang, Li-Hong; Shen, Guang-Mao; Wang, Jin-Jun

    2012-05-01

    Quantitative real time reverse transcriptase polymerase chain reaction (RT-qPCR) is preferred for gene expression analysis in living organisms. Currently, it is a valuable tool for biological and ecological studies as it provides a relatively straightforward way to assess the relevance of transcriptional regulation under developmental and stress tolerance conditions. However, studies have shown that some commonly used reference genes varied among different experimental treatments, thus, systematic evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of arthropods. The aim of this study is to identify the suitable reference genes for RT-qPCR experiments involving various developmental stages and/or under abiotic stresses in citrus red mite Panonychus citri, a key pest in citrus orchards worldwide. GeNorm, NormFinder, and Bestkeeper software analysis indicates that elongation factor-1 alpha (ELF1A), RNA polymerase II largest subunit, alpha tublin, and glyceraldhyde-3-phosphate dehydrogenase (GAPDH) are the most stable reference genes in various developmental stages, meanwhile, ELF1A and GAPDH were the most stable reference genes under various abiotic stresses. Furthermore, this study will serve as a resource to screen reference genes for gene expression studies in any other spider mite species. PMID:22203483

  3. Efficacy of laser capture microdissection plus RT-PCR technique in analyzing gene expression levels in human gastric cancer and colon cancer

    International Nuclear Information System (INIS)

    Thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase gene expressions are reported to be valid predictive markers for 5-fluorouracil sensitivity to gastrointestinal cancer. For more reliable predictability, their expressions in cancer cells and stromal cells in the cancerous tissue (cancerous stroma) have been separately investigated using laser capture microdissection. Thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase mRNA in cancer cells and cancerous stroma from samples of 47 gastric and 43 colon cancers were separately quantified by reverse transcription polymerase chain reaction after laser capture microdissection. In both gastric and colon cancers, thymidylate synthase and orotate phosphoribosyltransferase mRNA expressions were higher (p < 0.0001, p <0.0001 respectively in gastric cancer and P = 0.0002, p < 0.0001 respectively in colon cancer) and dihydropyrimidine dehydrogenase mRNA expressions were lower in cancer cells than in cancerous stroma (P = 0.0136 in gastric cancer and p < 0.0001 in colon cancer). In contrast, thymidine phosphorylase mRNA was higher in cancer cells than in cancerous stroma in gastric cancer (p < 0.0001) and lower in cancer cells than in cancerous stroma in colon cancer (P = 0.0055). By using this method, we could estimate gene expressions separately in cancer cells and stromal cells from colon and gastric cancers, in spite of the amount of stromal tissue. Our method is thought to be useful for accurately evaluating intratumoral gene expressions

  4. 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.

  5. Ghrelin-producing well-differentiated neuroendocrine tumor (carcinoid) of tailgut cyst. Morphological, immunohistochemical, ultrastructural, and RT-PCR study of a case and review of the literature.

    Science.gov (United States)

    La Rosa, Stefano; Boni, Luigi; Finzi, Giovanna; Vigetti, Davide; Papanikolaou, Nikolaos; Tenconi, Silvia Maria; Dionigi, Gianlorenzo; Clerici, Moira; Garancini, Silvana; Capella, Carlo

    2010-09-01

    Well-differentiated neuroendocrine tumors (carcinoids) arising in the presacral space are rare neoplasms that can arise in association with either sacrococcygeal teratomas or tailgut cysts. Although tumors arising in tailgut cysts are more frequent than those associated with teratomas, they are still very rare, and only 13 cases have been reported in the literature. We describe the first case of a carcinoid composed of ghrelin-producing cells arising in a tailgut cyst. Ghrelin production was demonstrated using immunohistochemistry, electron microscopy, and reverse transcription-polymerase chain reaction methods. A 73-year-old woman with back and pelvic pain was found to have a presacral mass histologically diagnosed, on needle biopsy, as a well-differentiated neuroendocrine tumor. Workup did not show another primary tumor or metastatic disease. The patient underwent laparoscopic resection of the mass, and the pathological diagnosis of the surgical specimen was of a tailgut cyst-associated carcinoid composed of ghrelin-producing cells. In addition, we have accurately reviewed the literature on presacral carcinoids, associated or unassociated with tailgut cysts, to give the reader a comprehensive overview of these very rare tumor types. PMID:20532674

  6. The effect of chain flexibility and chain mobility on radiation crosslinking reactions of polymers

    International Nuclear Information System (INIS)

    Flexibility of polymer chains is an important factor to effects of radiation crosslinking of the polymer. Polymers with flexible chains are easier to be crosslinked, with lower dose of gelation, than polymers with more rigid chains. And it is known that most polymers with abnormal rigidity can be radiation-crosslinked only at high temperatures when the molecular chains get enough mobility. The flexibility of polymer chains also influences the relationship between degree of degradation and radiation dose. A chain flexibility factor β has been introduced to modify the Charlesby-Pinner equation of sol-fraction and radiation dose. The new relationship equation applies to a wider range of polymers in radiation crosslinking. Studies also show that for flexible polymers with lower Tg and molecular internal rotating factor, mechanism of radiation crosslinking is mainly in H type, whereas for rigid polymers with higher Tg and molecular internal rotating factor, mechanism of radiation crosslinking is mainly in T type

  7. Expression of Kir2.1, SCN5a and SCN1b channel genes in mouse cardiomyocytes with various electric properties: patch clamp combined with single cell RT-PCR study%小鼠不同电特性的心肌细胞Kir2.1、SCN5a和SCN1b通道基因的表达:膜片钳结合单细胞RT-PCR技术研究

    Institute of Scientific and Technical Information of China (English)

    骆红艳; 梁华敏; 胡新武; 唐明

    2012-01-01

    This study is to explore a new method of investigating molecular basis for electrophysiological properties of early fetal car-diomyocytes. Single embryonic cardiomyocytes of mouse early developmental heart (E10.5) were obtained by a collagenase B digestion approach. After recording spontaneous action potential using whole cell patch clamp technique, the single cell was picked by a glass micropipette, followed by a standard RT-PCR to explore the expression levels of several ion channel genes. Three phenotypes of cardiomyocytes were demonstrated with distinct properties: ventricular-like, atrial-like, and pacemaker-like action potentials. Ventric-ular-like and atrial-like cells were characterized with much negative maximum diastolic potential (MDP) and a higher I/max (maximum velocity of depolarization) compared to pacemaker-like cells. MDP of ventricular-like cells was the most negative. In parallel, stronger expression of SCN5a, SCN1b and Kir2.1 were observed in ventricular-like and atrial-like cells compared to that of pacemaker-like cells, where Kir2.1 in ventricular-like cells was the most abundant. Cardiomyocytes with distinct electrophysiological properties had distinct gene expression pattern. Single cell RT-PCR combined with patch clamp technique could serve as a precise detector to analyze the molecular basis of the special electrophysiological characteristics of cardiomyocytes.%本文旨在应用膜片钳技术结合单细胞逆转录聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)技术研究小鼠胚胎心肌细胞的不同电生理学特点的分子生物学基础.应用胶原酶B消化分离得到小鼠胚胎早期(E10.5)单个心肌细胞,先用膜片钳技术记录单个胚胎心肌细胞的动作电位,随即应用玻璃微电极吸取该细胞内容物,进一步应用RT-PCR技术检测该单个心肌细胞离子通道基因Kir2.1、SCN5a和SCNlb的表达,以GAPDH为内参基因.全细胞电流钳技术记录到心室肌

  8. Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction.

    Science.gov (United States)

    Anindyajati; Artarini, A Anita; Riani, Catur; Retnoningrum, Debbie S

    2016-01-01

    Recombinant therapeutic proteins are biopharmaceutical products that develop rapidly for years. Recombinant protein production in certain hosts requires vector expression harboring the gene encoding the corresponding protein. Escherichia coli is the prokaryote organism mostly used in recombinant protein production, commonly using a plasmid as the expression vector. Recombinant protein production is affected by plasmid copy number harboring the encoded gene, hence the determination of plasmid copy number also plays an important role in establishing a recombinant protein production system. On the industrial scale, a low copy number of plasmids are more suitable due to their better stability. In the previous study we constructed pCAD, a plasmid derived from the low copy number pBR322 plasmid. This study was aimed to confirm pCAD's copy number by quantitative polymerase chain reaction (qPCR). Plasmid copy number was determined by comparing the quantification signal from the plasmid to those from the chromosome. Copy number was then calculated by using a known copy number plasmid as a standard. Two pairs of primers, called tdk and ori, were designed for targeting a single gene tdk in the chromosome and a conserved domain in the plasmid's ori, respectively. Primer quality was analyzed in silico using PrimerSelect DNASTAR and PraTo software prior to in vitro evaluation on primer specificity and efficiency as well as optimization of qPCR conditions. Plasmid copy number determination was conducted on E. coli lysates harboring each plasmid, with the number of cells ranging from 10(2)-10(5) cells/μL. Cells were lysed by incubation at 95ºC for 10 minutes, followed by immediate freezing at -4°C. pBR322 plasmid with the copy number of ~19 copies/cell was used as the standard, while pJExpress414-sod plasmid possessing the high copy number pUC ori was also determined to test the method being used. In silico analysis based on primer-primer and primer-template interactions showed

  9. DETERMINATION OF THE SPECTRUM OF ANTIBIOTIC RESISTANCE GENES HAVE PHENOTYPIC RESISTANT STRAINS OF PARIETAL INTESTINAL MICROBIOTA IN RATS BY RT-PCR

    Directory of Open Access Journals (Sweden)

    Bukina Y.V.

    2016-06-01

    Full Text Available Introduction. The problem of formation of bacterial resistance to glycopeptides and beta-lactam antibiotics (cephalosporins and carbapenems are used worldwide for the treatment of severe community acquired and nosocomial infections, especially caused by polymicrobial flora has become global and is a major factor limiting the effectiveness of antibiotic therapy. In this regard, the study of genetic microbial resistance determinants allows not only to carry out an effective antibiotic therapy, but also to identify two main processes leading to the development of epidemiologically significant events: the introduction of the agent in the risk population from the outside and in situ pathogen (spontaneous genetic drift targeted restructuring of the population. Therefore, the aim of our study was to investigate the resistance genes to carbapenems, cephalosporins, glycopeptides have clinically important phenotype of resistant strains of microorganisms families Enterobacteriaceae, Pseudomonadaceae, Bacteroidaceae, Enterococcaceae, Peptostreptococcaceae. Materials and methods. As a material for PCR studies 712 phenotypically resistant strains of microorganisms isolated from 80 rats "Wistar" line in microbiological study microflora of the wall were used. During the investigation 474 isolates of bacteria of the family Enterobacteriaceae, 39 - Pseudomonadaceae, 71 - Bacteroidaceae, 96 - Enterococcaceae, 32 - Peptostreptococcaceae were studied. Isolation of DNA from bacteria in the study was performed using reagents "DNA-Express" ("Litekh", Russia. For the detection of resistance genes by PCR in real time (RT-PCR reagent kits "FLUOROPOL-RV" ("Litekh", Russia were used. During the experiment, the VIM genes, OXA-48, NDM, KPC, responsible for the resistance of microorganisms to carbapenems, CTX-M - resistance to cephalosporins, as well as genes Van A and van B, the development of resistance to glycopeptides (vancomycin and teicoplanin were determined. Analysis

  10. Detection of Measles Virus Hemagglutinin Gene by RT-PCR%麻疹病毒H基因的特异性逆转录-聚合酶链反应检测

    Institute of Scientific and Technical Information of China (English)

    傅燕; 卢亦愚; 张严峻

    2001-01-01

    Using RT-PCR, we can detect measles virus hemagglutinin gene (H gene) of 635bp length directly from measles vaccine strain Shanghai-191, Edmonston strain, nasopharyngeal aspirates and throat swabs of the measles patients. The primers of RT-PCR based on H gene sequence of measles virus did not give the same products from rubella virus and influenza viruses (A1, A3, B). By second PCR amplification, the minimum quantity of measles virus required to give a positive signal was<0.01TCID50. Since the IgM antibody positive rate in early stage of measles patients is always low, the specificity and sensitivity of RT-PCR method may help diagnosing measles cases.%采用逆转录-聚合酶链反应(RT-PCR)直接从麻疹疫苗沪191株、Edmonston株以及临床麻疹患者的含漱液和咽拭子中检测出635bp的特异性H基因目的条带;而对风疹病毒和流行性感冒甲1型、甲3型、乙型病毒均未检测到相应条带。通过对RT-PCR产物的第2次扩增,可以使检测的灵敏度达到0.01TCID50。针对麻疹患者发病初期血清中IgM抗体阳性率偏低的特点,可以利用该方法的特异性和敏感性对临床麻疹病例作辅助处理。

  11. 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.

  12. The Construction and Clinic Application of RT-PCR Testing Process of Canine Distemper Virus%犬瘟热病毒RT-PCR检测方法的建立与临床应用

    Institute of Scientific and Technical Information of China (English)

    王金福; 张永霞; 顾剑新; 孙维平

    2009-01-01

    In recent years, the incidence of Canine Distemper (CD)disease increases obviously.Developing and perfecting CD diagnostic methods is in favor of guiding clinic therapy in time and is important to control the propagation of CD.According to the reported gene sequence of N protein of Canine Distemper Virus (CDV) in Gene Bank, a special pair of oligonucleotide primers were designed and using the extracted total RNA of CD vaccine strain as a template to set up RT-PCR testing process.Compared the clinic diagnosis effect of RT-PCR testing process with CDV antigen rapid diagnosis test paper,it showed that RT-PCR testing process has good sensitivity and specificity and was adapt to early rapid diagnosis and epidemiological investigation of CDV.%犬瘟热(CD)发病率近年来在我国呈明显上升趋势,对早期CD诊断方法的发展和完善,有利于指导临床及时治疗,对于控制CD的蔓延有重要意义.根据GenBank中CDV核衣壳蛋白(N)基因序列,设计合成一对特异性寡聚核苷酸引物,提取CDV疫苗株的总RNA进行反转录和PCR,建立RT-PCR检测方法,并应用于临床病例的诊断,同时和CDV抗原快速诊断试纸进行比较.结果显示:RT-PCR有很好的敏感性和特异性,适合对CDV进行早期快速诊断和流行病学调查.

  13. A novel technique used in the RT-PCR of single neurons%一种新型单个神经细胞的RT-PCR技术研究

    Institute of Scientific and Technical Information of China (English)

    高军峰; 侯保华; 廖立平; 陈恒玲; 李臣鸿

    2014-01-01

    It is very difficult to complete the application of RT-PCR based on a single cell because of the low level of mRNA.This study is to explore a novel experimental technique to achieve a repeatable process of the single cell RT-PCR,which could be used to characterize the biophysical properties of ion channels genes in a single neuron. The single cell was picked by a glass micropipette,followed by a standard RT-PCR to explore the expression levels of ion channel genes.Single-cell RT-PCR could serve as a precise detector to analyze the molecular basis of the special electrophysiological characteristics. Using the method proposed in the article can be repeated to obtain the expression of HCN1 and HPRT in the big cell of rat dorsal root ganglion. It is enough to continues the next experiment,which proves the feasibility of this technique.%提出一种新型可重复实现单细胞的RT-PCR的实验技术,扩增目的基因。应用玻璃微电极提取单个神经细胞,裂解获得mRNA,利用RT-PCR技术检测单个神经细胞的离子通道等基因的表达。应用此技术可以灵敏地检测特定电生理现象的分子生物学基础。此法可以比较大鼠背根神经节中大细胞内 HCN1的mRNA表达量。实验结果表明:扩增产物的量足够用于后续实验,而且3个重复的均一性也很好,充分表明了该研究提出的实验技术的可行性。

  14. RT-PCR在我院手足口病检测中的应用%Application of RT-PCR in Our Hospital in the Detection of Hand Foot and Mouth Disease

    Institute of Scientific and Technical Information of China (English)

    黄宏兰

    2014-01-01

    Objective To explore the RT-PCR technique the accuracy of the detection of hand, foot and mouth disease, provide reference for clinical monitoring. Methods Select our hospital outpatient service revenue of 200 cases of children with hand, foot and mouth disease as the research object, randomly selected from the gut, anus, throat swab specimens of 320 cases. Using RT-PCR technique to the qualitative detection of samples.Rusults Positive results in this study detected EV71 virus in 105 cases, 51 cases CoxA16 positive virus, EV positive for 11 cases of the virus. Two kinds of fluorescence rt-pcr detection result comparison, there was no statistical y significant difference ( > 0.05). Conclusion RT-PCR technique applied to clinical detection plays an important role in the hand, foot and mouth disease is worth in clinical promotion and use.%目的:探究RT-PCR技术检测手足口病的准确性,为后期临床监测提供参考。方法选取我院门诊收入的200例手足口病患儿作为研究对象,随机选取其肠道、肛门、咽拭子样本共320例。使用RT-PCR技术对样本进行定性检测,观察检测结果并进行分析。结果本研究中检出EV71阳性病毒105例,CoxA16阳性病毒51例,EV阳性病毒11例。两种荧光RT-PCR检测技术结果比较,差异无统计学意义(>0.05)。结论 RT-PCR技术应用于临床检测手足口病具有重要作用,值得在后期临床中推广及使用。

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

    OpenAIRE

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

    2007-01-01

    A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RN...

  16. 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.

  17. Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris

    Directory of Open Access Journals (Sweden)

    Biffali Elio

    2009-07-01

    Full Text Available Abstract Background Quantitative real-time polymerase chain reaction (RT-qPCR is valuable for studying the molecular events underlying physiological and behavioral phenomena. Normalization of real-time PCR data is critical for a reliable mRNA quantification. Here we identify reference genes to be utilized in RT-qPCR experiments to normalize and monitor the expression of target genes in the brain of the cephalopod mollusc Octopus vulgaris, an invertebrate. Such an approach is novel for this taxon and of advantage in future experiments given the complexity of the behavioral repertoire of this species when compared with its relatively simple neural organization. Results We chose 16S, and 18S rRNA, actB, EEF1A, tubA and ubi as candidate reference genes (housekeeping genes, HKG. The expression of 16S and 18S was highly variable and did not meet the requirements of candidate HKG. The expression of the other genes was almost stable and uniform among samples. We analyzed the expression of HKG into two different set of animals using tissues taken from the central nervous system (brain parts and mantle (here considered as control tissue by BestKeeper, geNorm and NormFinder. We found that HKG expressions differed considerably with respect to brain area and octopus samples in an HKG-specific manner. However, when the mantle is treated as control tissue and the entire central nervous system is considered, NormFinder revealed tubA and ubi as the most suitable HKG pair. These two genes were utilized to evaluate the relative expression of the genes FoxP, creb, dat and TH in O. vulgaris. Conclusion We analyzed the expression profiles of some genes here identified for O. vulgaris by applying RT-qPCR analysis for the first time in cephalopods. We validated candidate reference genes and found the expression of ubi and tubA to be the most appropriate to evaluate the expression of target genes in the brain of different octopuses. Our results also underline the

  18. Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system.

    Science.gov (United States)

    Carossino, Mariano; Lee, Pei-Yu A; Nam, Bora; Skillman, Ashley; Shuck, Kathleen M; Timoney, Peter J; Tsai, Yun-Long; Ma, Li-Juan; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Balasuriya, Udeni B R

    2016-08-01

    Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies. PMID:27036504

  19. Polymerase chain reaction for the diagnosis of viral hepatitis B and C.

    OpenAIRE

    Bréchot, C

    1993-01-01

    Polymerase chain reaction is a highly sensitive technique for the detection of hepatitis B virus-DNA and hepatitis C virus-RNA in serum, liver tissue, and peripheral mononuclear blood cells. In chronic hepatitis B, it is particularly useful for identification of infectious subjects who are hepatitis B surface antigen positive and anti-hepatitis B e antigen antibody-positive, and for follow up of hepatitis B virus infections in liver transplantation programmes. Polymerase chain reaction detect...

  20. Polymerase chain reaction for detection of the cholera enterotoxin operon of Vibrio cholerae.

    OpenAIRE

    1991-01-01

    We report a set of oligonucleotide primers and amplification conditions for the polymerase chain reaction to detect the ctx operon of Vibrio cholerae. The results of this assay on strains of V. cholerae and related organisms were identical with those obtained by the DNA colony hybridization test with the polynucleotide probe. The detection limit of this system was 1 pg of chromosomal DNA or broth culture containing three viable cells. The polymerase chain reaction method could directly detect...