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Sample records for quantitative real-time pcr

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

    Science.gov (United States)

    Wagner, Elke M

    2013-01-01

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

  2. Comparison of conventional culture and real-time quantitative PCR ...

    African Journals Online (AJOL)

    2009-10-28

    Oct 28, 2009 ... Results of real-time PCR were compared to con- ventional analysis .... water bath (Selecta 40W power, 40 kHz ultrasound fre- quency). ..... mental distributions of Legionella strains in France are different. J. clin. Microbiol.

  3. Diagnosis of aerobic vaginitis by quantitative real-time PCR.

    Science.gov (United States)

    Rumyantseva, T A; Bellen, G; Savochkina, Y A; Guschin, A E; Donders, G G G

    2016-07-01

    To evaluate a real-time PCR-based technique to quantify bacteria associated with aerobic vaginitis (AV) as a potential test. Vaginal samples from 100 women were tested by wet-mount microscopy, gram stain and quantitative real-time PCR targeting Enterobacteriacea, Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli, Streptococcus agalactiae, S. aureus; Lactobacillus spp. AV diagnosis obtained by wet-mount microscopy was used as reference. Some level of AV was diagnosed in 23 (23.7 %) cases. Various concentrations of Enterobacteriacea, Staphylococcus spp., Streptococcus spp. were detected an all patients. Enterococcus spp. were detected in 76 (78.3 %) cases. Summarized concentrations of aerobes were tenfold higher in AV-positive compared to AV-negative cases [7.30lg vs 6.06lg (p = 0.02)]. Concentrations of aerobes in severe, moderate and light AV cases did not vary significantly (p = 0.14). Concentration of lactobacilli was 1000-fold lower in AV-positive cases compared to normal cases (5.3lg vs 8.3lg, p < 0.0001). Streptococcus spp. dominated in the majority of AV-positive cases [19/22 (86.4 %) samples]. The relation of high loads of aerobes to the low numbers of Lactobacilli are a reliable marker for the presence of AV and could substitute microscopy as a test. PCR may be a good standardized substitution for AV diagnosis in settings where well-trained microscopists are lacking.

  4. Quantitative Detection of Respiratory Chlamydia pneumoniae Infection by Real-Time PCR

    OpenAIRE

    Kuoppa, Yvonne; Boman, Jens; Scott, Lena; Kumlin, Urban; Eriksson, Iréne; Allard, Annika

    2002-01-01

    Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.

  5. [Research progress of real-time quantitative PCR method for group A rotavirus detection].

    Science.gov (United States)

    Guo, Yan-Qing; Li, Dan-Di; Duan, Zhao-Jun

    2013-11-01

    Group A rotavirus is one of the most significant etiological agents which causes acute gastroenteritis among infants and young children worldwide. So far, several method which includes electron microscopy (EM), enzyme immunoassay (EIA), reverse transcription-polymerase chain reaction (RT-PCR)and Real-time Quantitative PCR has been established for the detection of rotavirus. Compared with other methods, Real-time quantitative PCR have advantages in specificity, sensitivity, genotyping and quantitative accuracy. This article shows a overview of the application of real-time quantitative PCR technique to detecte group A rotavirus.

  6. Digital PCR dynamic range is approaching that of real-time quantitative PCR.

    Science.gov (United States)

    Jones, Gerwyn M; Busby, Eloise; Garson, Jeremy A; Grant, Paul R; Nastouli, Eleni; Devonshire, Alison S; Whale, Alexandra S

    2016-12-01

    Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and KRAS genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR.

  7. Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR.

    NARCIS (Netherlands)

    Pierik, A.; Moamfa, M; van Zelst, M.; Clout, D.; Stapert, H.; Dijksman, Johan Frederik; Broer, D.; Wimberger-Friedl, R.

    2012-01-01

    Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that

  8. Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.

    Science.gov (United States)

    Jawhari, Maan; Abrahamian, Peter; Sater, Ali Abdel; Sobh, Hana; Tawidian, Patil; Abou-Jawdah, Yusuf

    2015-02-01

    Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.

  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. Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus

    OpenAIRE

    Kai Wang; Yi Niu; Qijun Wang; Haili Liu; Yi Jin; Shenglin Zhang

    2017-01-01

    Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used a...

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

    Science.gov (United States)

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

    2002-06-01

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

  12. Quantitative assay of photoinduced DNA strand breaks by real-time PCR.

    Science.gov (United States)

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-05

    Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies.

  13. Legionellosis and Lung Abscesses: Contribution of Legionella Quantitative Real-Time PCR to an Adapted Followup

    Directory of Open Access Journals (Sweden)

    G. Descours

    2013-01-01

    Full Text Available We report a case of severe Legionnaires' disease (LD complicated by a lung abscess in an immunocompetent patient who required ECMO therapy and thoracic surgery. The results of repeated Legionella quantitative real-time PCR performed on both sera and respiratory samples correlated with the LD severity and the poor clinical outcome. Moreover, the PCR allowed for the detection of Legionella DNA in the lung abscess specimen, which was negative when cultured for Legionella. This case report provides a logical basis for further investigations to examine whether the Legionella quantitative PCR could improve the assessment of LD severity and constitute a prognostic marker.

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

    Science.gov (United States)

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

    2016-01-01

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

  15. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    Science.gov (United States)

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  16. Embryonation of Ostertagia ostertagi eggs affects the outcome of real-time quantitative PCR

    DEFF Research Database (Denmark)

    Drag, Markus; Höglund, Johan; Nejsum, Peter

    prior to detection and quantification by real-time quantitative polymerase chain reaction (qPCR). Fresh O. ostertagi eggs were isolated from cattle faeces and stored at 4°C or 25°C under aerobic or anaerobic conditions. Embryonation was monitored by microscopy and the ITS2 copies were determined by q...... the outcome of qPCR analysis for the quantitative determination of O. ostertagi eggs in cattle faeces. Cold storage at 4°C for up to 3 days or anaerobicvacuum packing at 25°C for up to 336 h will entail no undesirable effects on ITS2 copies....

  17. Embryonation of Ostertagia ostertagi eggs affects the outcome of real-time quantitative PCR

    DEFF Research Database (Denmark)

    Drag, Markus; Höglund, Johan; Nejsum, Peter

    prior to detection and quantification by real-time quantitative polymerase chain reaction (qPCR) . Fresh O. ostertagi eggs were isolated from cattle faeces and stored at 4°C or 25°C under aerobic or anaerobic conditions. Embryonation was monitored by microscopy and the ITS2 copies were determined by q...... the outcome of qPCR analysis for the quantitative determination of O. ostertagi eggs in cattle faeces. Cold storage at 4°C for up to 3 days or anaerobic vacuum packing at 25°C for up to 336 h will entail no undesirable effects on ITS2 copies....

  18. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

    DEFF Research Database (Denmark)

    Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

    2004-01-01

    We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods....... Study cases (27 carcinomas and 3 ductal breast carcinoma in situ (DCIS) cases) showed varying Her-2 expression as determined by IHC (HercepTest). In carcinomas, there was a good correlation between HER-2 DNA amplification and strong HER-2 protein expression detected by FISH and IHC, respectively....... A single DCIS case was amplified in FISH, but not in IHC. Both HER-2 gene amplification and expression could be quantified in microdissected paraffin-embedded tumors using real-time PCR, DNA and RNA being successfully detected in 146 of 150 (97%) and 141 of 150 (94%) samples, respectively. PCR analysis...

  19. Detection of Leishmania infantum DNA in conjunctival swabs of cats by quantitative real-time PCR.

    Science.gov (United States)

    Benassi, Julia Cristina; Benvenga, Graziella U; Ferreira, Helena Lage; Pereira, Vanessa F; Keid, Lara B; Soares, Rodrigo; Oliveira, Tricia Maria Ferreira de Sousa

    2017-06-01

    Although some studies have investigated the potential role of cats as a reservoir for Leishmania, their role in the epidemiology of visceral leishmaniasis (VL) is still poorly understood. Molecular diagnostic techniques are an important tool in VL diagnosis, and PCR shows high sensitivity and specificity for Leishmania spp. detection. Quantitative real-time PCR (qPCR) is a method that permits quantitative analysis of a large number of samples, resulting in more sensitive, accurate, and reproducible measurements of specific DNA present in the sample. This study compared real-time PCR (qPCR) and conventional PCR (cPCR) for detection of Leishmania spp. in blood and conjunctival swab (CS) samples of healthy cats from a non-endemic area in the state of São Paulo, Brazil. Of all CS samples, 1.85% (2/108) were positive for Leishmania spp. by both cPCR as qPCR (kappa index = 1), indicating excellent agreement between the two methods. The DNA from the two CS-cPCR- and CS-qPCR-positive samples was further tested with a PCR test amplifying the Leishmania spp. discriminative rRNA internal transcribed spacer 1 (ITS 1), of which one sample generated a 300-350-bp DNA fragment whose size varies according to the Leishmania species. Following sequencing, the fragment showed 100% similarity to a GenBank L. infantum sequence obtained from a cat in Italy. In conclusion, the association of qPCR and CS proved to be effective for detection of Leishmania in cats. Conjunctival swab samples were shown to be a practical and better alternative to blood samples and may be useful in the diagnosis and studies of feline leishmaniasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Development of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    Science.gov (United States)

    Oguchi, Taichi; Onishi, Mari; Minegishi, Yasutaka; Kurosawa, Yasunori; Kasahara, Masaki; Akiyama, Hiroshi; Teshima, Reiko; Futo, Satoshi; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2009-06-01

    A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this.

  1. Quantitation of HIV-1 RNA in breast milk by real time PCR.

    Science.gov (United States)

    Becquart, Pierre; Foulongne, Vincent; Willumsen, Juana; Rouzioux, Christine; Segondy, Michel; Van de Perre, Philippe

    2006-04-01

    HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.

  2. Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability.

    Science.gov (United States)

    Hilscher, Chelsey; Vahrson, Wolfgang; Dittmer, Dirk P

    2005-11-27

    Quantitative real-time PCR has become the method of choice for measuring mRNA transcription. Recently, fast PCR protocols have been developed as a means to increase assay throughput. Yet it is unclear whether more rapid cycling conditions preserve the original assay performance characteristics. We compared 16 primer sets directed against Epstein-Barr virus (EBV) mRNAs using universal and fast PCR cycling conditions. These primers are of clinical relevance, since they can be used to monitor viral oncogene and drug-resistance gene expression in transplant patients and EBV-associated cancers. While none of the primers failed under fast PCR conditions, the fast PCR protocols performed worse than universal cycling conditions. Fast PCR was associated with a loss of sensitivity as well as higher variability, but not with a loss of specificity or with a higher false positive rate.

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

    OpenAIRE

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

    2007-01-01

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

  4. Design of primers and probes for quantitative real-time PCR methods.

    Science.gov (United States)

    Rodríguez, Alicia; Rodríguez, Mar; Córdoba, Juan J; Andrade, María J

    2015-01-01

    Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR(®) Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR). This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.

  5. Reliability of quantitative real-time PCR for bacterial detection in cystic fibrosis airway specimens.

    Directory of Open Access Journals (Sweden)

    Edith T Zemanick

    Full Text Available The cystic fibrosis (CF airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities≥10(2 rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.

  6. Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize.

    Science.gov (United States)

    Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Chen, Xiaoyun; Wang, Qiang; Xu, Junfeng

    2016-12-01

    This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

  7. 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer

    Directory of Open Access Journals (Sweden)

    Dráberová Lubica

    2011-04-01

    Full Text Available Abstract Background Quantitative real-time PCR (qPCR is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI. In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify. Results We found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer. These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons. Conclusions The combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty.

  8. The use sof real-time quantitative PCR for the analysis of cytokine mRNA levels

    NARCIS (Netherlands)

    Forlenza, M.; Kaiser, T.; Savelkoul, H.F.J.; Wiegertjes, G.F.

    2012-01-01

    Over the last decade, real-time-quantitative PCR (RT-qPCR) analysis has become the method of choice not only for quantitative and accurate measurement of mRNA expression levels, but also for sensitive detection of rare or mutated DNA species in diagnostic research. RT-qPCR is based on the standard p

  9. Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Van Zeveren Alex

    2005-12-01

    Full Text Available Abstract Background Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. Results In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. Conclusion Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

  10. Validation of absolute quantitative real-time PCR for the diagnosis of Streptococcus agalactiae in fish.

    Science.gov (United States)

    Sebastião, Fernanda de A; Lemos, Eliana G M; Pilarski, Fabiana

    2015-12-01

    Streptococcus agalactiae (GBS) are Gram-positive cocci responsible for substantial losses in tilapia fish farms in Brazil and worldwide. It causes septicemia, meningoencephalitis and mortality of whole shoals that can occur within 72 h. Thus, diagnostic methods are needed that are rapid, specific and sensitive. In this study, a pair of specific primers for GBS was generated based on the cfb gene sequence and initially evaluated by conventional PCR. The protocols for absolute quantitative real-time PCR (qPCR) were then adapted to validate the technique for the identification and quantification of GBS isolated by real-time detection of amplicons using fluorescence measurements. Finally, an infectivity test was conducted in tilapia infected with GBS strains. Total DNA from the host brain was subjected to the same technique, and the strains were re-isolated to validate Koch's postulates. The assay showed 100% specificity for the other bacterial species evaluated and a sensitivity of 367 gene copies per 20 mg of brain tissue within 4 h, making this test a valuable tool for health monitoring programs.

  11. Quantitative detection of Clostridium tyrobutyricum in milk by real-time PCR.

    Science.gov (United States)

    López-Enríquez, Lorena; Rodríguez-Lázaro, David; Hernández, Marta

    2007-06-01

    We developed a real-time PCR assay for the quantitative detection of Clostridium tyrobutyricum, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (R(2) > 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300 C. tyrobutyricum spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.

  12. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    Science.gov (United States)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  13. Evaluation of postmortem bacterial migration using culturing and real-time quantitative PCR.

    Science.gov (United States)

    Tuomisto, Sari; Karhunen, Pekka J; Vuento, Risto; Aittoniemi, Janne; Pessi, Tanja

    2013-07-01

    Postmortem bacteriology can be a valuable tool for evaluating deaths due to bacterial infection or for researching the involvement of bacteria in various diseases. In this study, time-dependent postmortem bacterial migration into liver, mesenteric lymph node, pericardial fluid, portal, and peripheral vein was analyzed in 33 autopsy cases by bacterial culturing and real-time quantitative polymerase chain reaction (RT-qPCR). None suffered or died from bacterial infection. According to culturing, pericardial fluid and liver were the most sterile samples up to 5 days postmortem. In these samples, multigrowth and staphylococci were not or rarely detected. RT-qPCR was more sensitive and showed higher bacterial positivity in all samples. Relative amounts of intestinal bacterial DNA (bifidobacteria, bacteroides, enterobacter, clostridia) increased with time. Sterility of blood samples was low during the studied time periods (1-7 days). The best postmortem microbiological sampling sites were pericardial fluid and liver up to 5 days after death.

  14. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    Science.gov (United States)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-05-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.

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

    Directory of Open Access Journals (Sweden)

    Dongari-Bagtzoglou Anna

    2011-05-01

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

  16. Hygienization by anaerobic digestion: comparison between evaluation by cultivation and quantitative real-time PCR.

    Science.gov (United States)

    Lebuhn, M; Effenberger, M; Garcés, G; Gronauer, A; Wilderer, P A

    2005-01-01

    In order to assess hygienization by anaerobic digestion, a comparison between evaluation by cultivation and quantitative real-time PCR (qPCR) including optimized DNA extraction and quantification was carried out for samples from a full-scale fermenter cascade (F1, mesophilic; F2, thermophilic; F3, mesophilic). The system was highly effective in inactivating (pathogenic) viable microorganisms, except for spore-formers. Conventionally performed cultivation underestimated viable organisms particularly in F2 and F3 by a factor of at least 10 as shown by data from extended incubation times, probably due to the rise of sublethally injured (active but not cultivable) cells. Incubation should hence be extended adequately in incubation-based hygiene monitoring of stressed samples, in order to minimize contamination risks. Although results from qPCR and cultivation agreed for the equilibrated compartments, considerably higher qPCR values were obtained for the fermenters. The difference probably corresponded to DNA copies from decayed cells that had not yet been degraded by the residual microbial activity. An extrapolation from qPCR determination to the quantity of viable organisms is hence not justified for samples that had been exposed to lethal stress.

  17. RDML: structured language and reporting guidelines for real-time quantitative PCR data

    NARCIS (Netherlands)

    Lefever, S.; Hellemans, J.; Pattyn, F.; Przybylski, D.R.; Taylor, C.; Geurts, R.; Untergasser, A.; Vandesompele, J.

    2009-01-01

    The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium (http://www.rdml.org) to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and be

  18. Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR

    Directory of Open Access Journals (Sweden)

    López-Revilla Rubén

    2010-05-01

    Full Text Available Abstract Background We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR in the presence of the DNA intercalating agent EvaGreen. Results Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp-long insert of the human papillomavirus type 16 (HPV16 E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 102-2.5 × 106 initial pHV101 copies had threshold cycle (Ct values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C. Conclusions Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.

  19. Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

    Institute of Scientific and Technical Information of China (English)

    HE Xiu-ting; LIU Cheng-cheng; LI Zhao-qun; ZHANG Zan; LI Guo-qing; LI Fei; DONG Shuang-lin

    2014-01-01

    The normalization of quantitative real-time PCR (qPCR) is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions. qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, ifve new housekeeping genes (LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9) in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable thanβ-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantiifcation. These results were further conifrmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantiifcation by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 orβ-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replaceβ-actin as the reference genes for qPCR in L. striatellus.

  20. Assessing the performance capabilities of LRE-based assays for absolute quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Robert G Rutledge

    potential to fundamentally transform how real-time qPCR is conducted.

  1. Development of real time PCR for detection and quantitation of Dengue Viruses

    Directory of Open Access Journals (Sweden)

    Singh A

    2009-01-01

    Full Text Available Abstract Background Dengue virus (DENV, a mosquito borne flavivirus is an important pathogen causing more than 50 million infections every year around the world. Dengue diagnosis depends on serology, which is not useful in the early phase of the disease and virus isolation, which is laborious and time consuming. There is need for a rapid, sensitive and high throughput method for detection of DENV in the early stages of the disease. Several real-time PCR assays have been described for dengue viruses, but there is scope for improvement. The new generation TaqMan Minor Groove Binding (MGB probe approach was used to develop an improved real time RT-PCR (qRT-PCR for DENV in this study. Results The 3'UTR of thirteen Indian strains of DENV was sequenced and aligned with 41 representative sequences from GenBank. A region conserved in all four serotypes was used to target primers and probes for the qRT-PCR. A single MGB probe and a single primer pair for all the four serotypes of DENV were designed. The sensitivity of the two step qRT-PCR assay was10 copies of RNA molecules per reaction. The specificity and sensitivity of the assay was 100% when tested with a panel of 39 known positive and negative samples. Viral RNA could be detected and quantitated in infected mouse brain, cell cultures, mosquitoes and clinical samples. Viral RNA could be detected in patients even after seroconversion till 10 days post onset of infection. There was no signal with Japanese Encephalitis (JE, West Nile (WN, Chikungunya (CHK viruses or with Leptospira, Plasmodium vivax, Plasmodium falciparum and Rickettsia positive clinical samples. Conclusion We have developed a highly sensitive and specific qRT-PCR for detection and quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia and leptospira occur. The

  2. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Quantitative analysis of the dystrophin gene by real-time PCR

    Directory of Open Access Journals (Sweden)

    Maksimovic Nela

    2012-01-01

    Full Text Available Duchenne and Becker muscular dystrophy (DMD/BMD are severe X-linked neuromuscular disorders caused by mutations in the dystrophin gene. Our aim was to optimize a quantitative real-time PCR method based on SYBR® Green I chemistry for routine diagnostics of DMD/BMD deletion carriers. Twenty female relatives of DMD/BMD patients with previously detected partial gene deletions were studied. The relative quantity of the target exons was calculated by a comparative threshold cycle method (ΔΔCt. The carrier status of all subjects was successfully determined. The gene dosage ratio for non-carriers was 1.07±0.20, and for carriers 0.56±0.11. This assay proved to be simple, rapid, reliable and cost-effective.

  5. Investigations on abundance and activity of microbial sponge symbionts using quantitative real - time PCR

    DEFF Research Database (Denmark)

    Kumala, Lars; Hentschel, Ute; Bayer, Kristina

    Marine sponges are hosts to dense and diverse microbial consortia that are likely to play a key role in the metabolic processes of the host sponge due to their enormous abundance. Common symbioses between nitrogen transforming microorganisms and sponges indicate complex nitrogen cycling within...... the host. Of particular interest is determining the community structure and function of microbial symbionts in order to gain deeper insight into host-symbiont interactions. We investigated the abundance and activity of microbial symbionts in two Mediterranean sponge species using quantitative real-time PCR....... An absolute quantification of functional genes and transcripts in archaeal and bacterial symbionts was conducted to determine their involvement in nitrification and denitrification, comparing the low microbial abundance (LMA) sponge Dysidea avara with the high microbial abundance (HMA) representative Aplysina...

  6. A method for accurate detection of genomic microdeletions using real-time quantitative PCR

    Directory of Open Access Journals (Sweden)

    Bassett Anne S

    2005-12-01

    Full Text Available Abstract Background Quantitative Polymerase Chain Reaction (qPCR is a well-established method for quantifying levels of gene expression, but has not been routinely applied to the detection of constitutional copy number alterations of human genomic DNA. Microdeletions or microduplications of the human genome are associated with a variety of genetic disorders. Although, clinical laboratories routinely use fluorescence in situ hybridization (FISH to identify such cryptic genomic alterations, there remains a significant number of individuals in which constitutional genomic imbalance is suspected, based on clinical parameters, but cannot be readily detected using current cytogenetic techniques. Results In this study, a novel application for real-time qPCR is presented that can be used to reproducibly detect chromosomal microdeletions and microduplications. This approach was applied to DNA from a series of patient samples and controls to validate genomic copy number alteration at cytoband 22q11. The study group comprised 12 patients with clinical symptoms of chromosome 22q11 deletion syndrome (22q11DS, 1 patient trisomic for 22q11 and 4 normal controls. 6 of the patients (group 1 had known hemizygous deletions, as detected by standard diagnostic FISH, whilst the remaining 6 patients (group 2 were classified as 22q11DS negative using the clinical FISH assay. Screening of the patients and controls with a set of 10 real time qPCR primers, spanning the 22q11.2-deleted region and flanking sequence, confirmed the FISH assay results for all patients with 100% concordance. Moreover, this qPCR enabled a refinement of the region of deletion at 22q11. Analysis of DNA from chromosome 22 trisomic sample demonstrated genomic duplication within 22q11. Conclusion In this paper we present a qPCR approach for the detection of chromosomal microdeletions and microduplications. The strategic use of in silico modelling for qPCR primer design to avoid regions of repetitive

  7. Quantitative detection of hazelnut (Corylus avellana) in cookies: ELISA versus real-time PCR.

    Science.gov (United States)

    Platteau, Céline; De Loose, Marc; De Meulenaer, Bruno; Taverniers, Isabel

    2011-11-09

    Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.

  8. Influence of segmenting fluids on efficiency, crossing point and fluorescence level in real time quantitative PCR.

    Science.gov (United States)

    Walsh, E J; King, C; Grimes, R; Gonzalez, A

    2006-03-01

    The two-phase segmented flow approach to the processing and quantitative analysis of biological samples in microdevices offers significant advantages over the single-phase continuous flow methodology. Despite this, little is known about the compatibility of samples and reactants with segmenting fluids, although a number of investigators have reported reduced yield and inhibition of enzymatic reactions depending on the segmenting fluid employed. The current study addresses the compatibility of various segmenting fluids with real time quantitative PCR to understand the physicochemical requirements of this important reaction in biotechnology. The results demonstrate that creating a static segmenting fluid/PCR mix interface has a negligible impact on the reaction efficiency, crossing threshold and end fluorescence levels using a variety of segmenting fluids. The implication is then that the previously reported inhibitory effects are the result of the dynamic motion between the segmenting fluid and the sample in continuously flowing systems. The results presented here are a first step towards understanding the limitations of the segmented flow methodology, which are necessary to bring this approach into mainstream use.

  9. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay

    Directory of Open Access Journals (Sweden)

    Liu Cindy M

    2012-11-01

    Full Text Available Abstract Background Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. Methods We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE guidelines. Results We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant’s is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from Conclusions FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.

  10. Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae.

    Science.gov (United States)

    Takahashi, Hajime; Saito, Rumi; Miya, Satoko; Tanaka, Yuichiro; Miyamura, Natsumi; Kuda, Takashi; Kimura, Bon

    2017-04-04

    The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A real-time PCR and culture methods was obtained in a majority of the food samples (81.8%), with good correlation (r(2)=0.8285). This study demonstrated that the rplP-targeted real-time PCR method could detect and enumerate Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods.

  11. Reference gene selection for quantitative real-time PCR normalization in Quercus suber.

    Science.gov (United States)

    Marum, Liliana; Miguel, Andreia; Ricardo, Cândido P; Miguel, Célia

    2012-01-01

    The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

  12. Reference gene selection for quantitative real-time PCR normalization in Quercus suber.

    Directory of Open Access Journals (Sweden)

    Liliana Marum

    Full Text Available The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber, have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old or collected in different dates (active growth period versus dormancy. The three statistical methods (geNorm, NormFinder, and CV method used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

  13. Easy-to-use strategy for reference gene selection in quantitative real-time PCR experiments.

    Science.gov (United States)

    Klenke, Stefanie; Renckhoff, Kristina; Engler, Andrea; Peters, Jürgen; Frey, Ulrich H

    2016-12-01

    Real-time PCR is an indispensable technique for mRNA expression analysis but conclusions depend on appropriate reference gene selection. However, while reference gene selection has been a topic of publications, this issue is often disregarded when measuring target mRNA expression. Therefore, we (1) evaluated the frequency of appropriate reference gene selection, (2) suggest an easy-to-use tool for least variability reference gene selection, (3) demonstrate application of this tool, and (4) show effects on target gene expression profiles. All 2015 published articles in Naunyn-Schmiedeberg's Archives of Pharmacology were screened for the use of quantitative real-time PCR analysis and selection of reference genes. Target gene expression (Vegfa, Grk2, Sirt4, and Timp3) in H9c2 cells was analyzed following various interventions (hypoxia, hyperglycemia, and/or isoflurane exposure with and without subsequent hypoxia) in relation to putative reference genes (Actb, Gapdh, B2m, Sdha, and Rplp1) using the least variability method vs. an arbitrarily selected but established reference gene. In the vast majority (18 of 21) of papers, no information was provided regarding selection of an appropriate reference gene. In only 1 of 21 papers, a method of appropriate reference gene selection was described and in 2 papers reference gene selection remains unclear. The method of reference gene selection had major impact on interpretation of target gene expression. With hypoxia, for instance, the least variability gene was Rplp1 and target gene expression (Vefga) heavily showed a 2-fold up-regulation (p = 0.022) but no change (p = 0.3) when arbitrarily using Gapdh. Frequency of appropriate reference gene selection in this journal is low, and we propose our strategy for reference gene selection as an easy tool for proper target gene expression.

  14. Quantitative Analysis of Epstein-Barr Virus Load by Using a Real-Time PCR Assay

    OpenAIRE

    1999-01-01

    To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 107 copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 103.7 copies/μg of DNA in patients with EBV-related lymphoprolif...

  15. [Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm, Helicoverna armigera].

    Science.gov (United States)

    Chandra, G Sharath; Asokan, R; Manamohan, M; Kumar, N K K; Sita, T

    2014-01-01

    Reverse-transcription quantitative real-time PCR (RT-qPCR), a sensitive technique is being extensively employed in quantification of gene expression. However this requires normalization with suitable reference gene (RG) which is crucial in minimizing inter sample variations. Information regarding suitable RG is scarce in general and more so in insects, including the cotton bollworm, Helicoverpa armigera, an economically important pest. In management of this pest RNA interference (RNAi), is perceived as a potential tool, which is achieved by double-stranded RNA (dsRNA) delivery. These studies demand accurate quantification of gene silencing. In this study we assessed the suitability of five RGs viz. β-actin (ACTB), 18S rRNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB) and elongation fator-1-alfa (EF1-α) for gene expression studies in dsRNA treatment and across different developmental stages of H. armigera and ranked using geNorm, NormFinder and BestKeeper software programs. Data analysis revealed that best ranked RGs were varied in dsRNA treatment and in developmental stages. Under dsRNA treatment, 18S and GAPDH were more stable whereas, TUB and GAPDH were more stable across developmental stages. We also demonstrate that inappropriate selection of RG led to erroneous estimation of the target gene, chymotrypsin, expression. These results facilitate accurate quantification of gene expression in H. armigera.

  16. Selection of Suitable Reference Genes for Quantitative Real-time PCR in Sapium sebiferum

    Directory of Open Access Journals (Sweden)

    Xue Chen

    2017-05-01

    Full Text Available Chinese tallow (Sapium sebiferum L. is a promising landscape and bioenergy plant. Measuring gene expression by quantitative real-time polymerase chain reaction (qRT-PCR can provide valuable information on gene function. Stably expressed reference genes for normalization are a prerequisite for ensuring the accuracy of the target gene expression level among different samples. However, the reference genes in Chinese tallow have not been systematically validated. In this study, 12 candidate reference genes (18S, GAPDH, UBQ, RPS15, SAND, TIP41, 60S, ACT7, PDF2, APT, TBP, and TUB were investigated with qRT-PCR in 18 samples, including those from different tissues, from plants treated with sucrose and cold stresses. The data were calculated with four common algorithms, geNorm, BestKeeper, NormFinder, and the delta cycle threshold (ΔCt. TIP41 and GAPDH were the most stable for the tissue-specific experiment, GAPDH and 60S for cold treatment, and GAPDH and UBQ for sucrose stresses, while the least stable genes were 60S, TIP41, and 18S respectively. The comprehensive results showed APT, GAPDH, and UBQ to be the top-ranked stable genes across all the samples. The stability of 60S was the lowest during all experiments. These selected reference genes were further validated by comparing the expression profiles of the chalcone synthase gene in Chinese tallow in different samples. The results will help to improve the accuracy of gene expression studies in Chinese tallow.

  17. The quantification of spermatozoa by real-time quantitative PCR, spectrophotometry, and spermatophore cap size.

    Science.gov (United States)

    Doyle, Jacqueline M; McCormick, Cory R; DeWoody, J Andrew

    2011-01-01

    Many animals, such as crustaceans, insects, and salamanders, package their sperm into spermatophores, and the number of spermatozoa contained in a spermatophore is relevant to studies of sexual selection and sperm competition. We used two molecular methods, real-time quantitative polymerase chain reaction (RT-qPCR) and spectrophotometry, to estimate sperm numbers from spermatophores. First, we designed gene-specific primers that produced a single amplicon in four species of ambystomatid salamanders. A standard curve generated from cloned amplicons revealed a strong positive relationship between template DNA quantity and cycle threshold, suggesting that RT-qPCR could be used to quantify sperm in a given sample. We then extracted DNA from multiple Ambystoma maculatum spermatophores, performed RT-qPCR on each sample, and estimated template copy numbers (i.e. sperm number) using the standard curve. Second, we used spectrophotometry to determine the number of sperm per spermatophore by measuring DNA concentration relative to the genome size. We documented a significant positive relationship between the estimates of sperm number based on RT-qPCR and those based on spectrophotometry. When these molecular estimates were compared to spermatophore cap size, which in principle could predict the number of sperm contained in the spermatophore, we also found a significant positive relationship between sperm number and spermatophore cap size. This linear model allows estimates of sperm number strictly from cap size, an approach which could greatly simplify the estimation of sperm number in future studies. These methods may help explain variation in fertilization success where sperm competition is mediated by sperm quantity.

  18. Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene

    NARCIS (Netherlands)

    Tobias, T.J.; Bouma, A.; Klinkenberg, D.; Daemen, A.J.J.M.; Stegeman, J.A.; Wagenaar, J.A.; Duim, B.

    2012-01-01

    A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected

  19. A human fecal contamination index for ranking impaired recreational watersusing the HF183 quantitative real-time PCR method

    Science.gov (United States)

    Human fecal pollution of surface water remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for recreational water quality risk managem...

  20. Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

    Science.gov (United States)

    There is a growing interest in the application of human-associated fecal sourceidentification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data q...

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

    Science.gov (United States)

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

  2. A human fecal contamination index for ranking impaired recreational watersusing the HF183 quantitative real-time PCR method

    Science.gov (United States)

    Human fecal pollution of surface water remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for recreational water quality risk managem...

  3. Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

    Science.gov (United States)

    There is a growing interest in the application of human-associated fecal sourceidentification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data q...

  4. Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene

    NARCIS (Netherlands)

    Tobias, T.J.; Bouma, A.; Klinkenberg, D.; Daemen, A.J.J.M.; Stegeman, J.A.; Wagenaar, J.A.; Duim, B.

    2012-01-01

    A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected convention

  5. Estimating marginal properties of quantitative real-time PCR data using nonlinear mixed models

    DEFF Research Database (Denmark)

    Gerhard, Daniel; Bremer, Melanie; Ritz, Christian

    2014-01-01

    A unified modeling framework based on a set of nonlinear mixed models is proposed for flexible modeling of gene expression in real-time PCR experiments. Focus is on estimating the marginal or population-based derived parameters: cycle thresholds and ΔΔc(t), but retaining the conditional mixed mod...

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

    Directory of Open Access Journals (Sweden)

    Meng Sun

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

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

    Science.gov (United States)

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

    2015-01-01

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

  8. TaqMan real-time PCR for detection and quantitation of squash leaf curl virus in cucurbits.

    Science.gov (United States)

    Kuan, Cheng-Ping; Huang, Hung-Chang; Chang, Chia-Che; Lu, Yi-Lin

    2012-02-01

    A real-time PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of the squash leaf curl virus (SLCV) in melon and squash plants. This method was highly specific to SLCV and it was about one thousand times more sensitive than the conventional PCR method. The protocol of the real-time PCR established in this study enabled detection of as little as 10(2) copies of SLCV DNA with CP gene as the target. This TaqMan real-time PCR assay for detection and quantitation of SLCV would be a useful tool for application in quarantine and certification of SLCV in cucurbits as well as in the research of disease resistance and epidemiology.

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

    Science.gov (United States)

    Painter, Janet E; Lambert, Kris N

    2003-03-01

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

  10. Microscopy, culture, and quantitative real-time PCR examination confirm internalization of mycobacteria in plants.

    Science.gov (United States)

    Kaevska, M; Lvoncik, S; Slana, I; Kulich, P; Kralik, P

    2014-07-01

    The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10(8) to 10(10) cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10(4) cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.

  11. Reference gene selection for quantitative real-time PCR normalization in Reaumuria soongorica.

    Science.gov (United States)

    Yan, Xia; Dong, Xicun; Zhang, Wen; Yin, Hengxia; Xiao, Honglang; Chen, Peng; Ma, Xiao-Fei

    2014-01-01

    Despite its superiority for evaluating gene expression, real-time quantitative polymerase chain reaction (qPCR) results can be significantly biased by the use of inappropriate reference genes under different experimental conditions. Reaumuria soongorica is a dominant species of desert ecosystems in arid central Asia. Given the increasing interest in ecological engineering and potential genetic resources for arid agronomy, it is important to analyze gene function. However, systematic evaluation of stable reference genes should be performed prior to such analyses. In this study, the stabilities of 10 candidate reference genes were analyzed under 4 kinds of abiotic stresses (drought, salt, dark, and heat) within 4 accessions (HG010, HG020, XGG030, and XGG040) from 2 different habitats using 3 algorithms (geNorm, NormFinder, and BestKeeper). After validation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large unite (rbcL) expression pattern, our data suggested that histone H2A (H2A) and eukaryotic initiation factor 4A-2 (EIF4A2) were the most stable reference genes, cyclophilin (CYCL) was moderate, and elongation factor 1α (EF1α) was the worst choice. This first systematic analysis for stably expressed genes will facilitate future functional analyses and deep mining of genetic resources in R. soongorica and other species of the Reaumuria genus.

  12. Studying the replication history of human B lymphocytes by real-time quantitative (RQ)-PCR.

    Science.gov (United States)

    van Zelm, Menno C; Berkowska, Magdalena A; van Dongen, Jacques J M

    2013-01-01

    The cells of the adaptive immune system, B and T lymphocytes, each generate a unique antigen receptor through V(D)J recombination of their immunoglobulin (Ig) and T cell receptor (TCR) loci, respectively. Such rearrangements join coding elements to form a coding joint and delete the intervening DNA as circular excision products containing the signal joint. These excision circles are stable structures that cannot replicate and have no function in the cell. Since the coding joint in the genome is replicated with each cell division, the ratio between coding joints and signal joints in a population of B cells can be used as a measure for proliferation. This chapter describes a real-time quantitative (RQ-)PCR-based approach to quantify proliferation through calculating the ratio between coding joints and signal joints of the frequently occurring intronRSS-Kde rearrangements in the IGK light chain locus. The approach is useful to study basic B-cell biology as well as abnormal proliferation in human diseases.

  13. To Normalize the Using of Quantitative Real-time Reverse Transcription PCR%Real time RT-qPCR 检测规范化

    Institute of Scientific and Technical Information of China (English)

    马俊彦; 林俊

    2010-01-01

    Real time RT-qPCR(real-time quantitatire reverse transcription-PCR)是一种快速、简便、准确、灵敏、成本低廉的基因检测技术,被认为是目前检测基因在转录水平表达的金标准.研究发现Real time RT-qPCR的检测结果会受到实验设计、引物、模板的质量、内参的选择及数据分析的方法等多个因素的影响,规范实验设计及操作是得到可靠结论的前提和必要条件.对近年来国内外涉及Real time RT-qPCR整个实验流程及数据发表的一些规范及标准进行综述,以便规范实验设计及操作,加强实验质量控制,促进研究结果的推广,和更好地发挥其应用价值.

  14. Estimating the number of integrations in transformed plants by quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Vaira Anna Maria

    2002-10-01

    Full Text Available Abstract Background When generating transformed plants, a first step in their characterization is to obtain, for each new line, an estimate of how many copies of the transgene have been integrated in the plant genome because this can deeply influence the level of transgene expression and the ease of stabilizing expression in following generations. This task is normally achieved by Southern analysis, a procedure that requires relatively large amounts of plant material and is both costly and labour-intensive. Moreover, in the presence of rearranged copies the estimates are not correct. New approaches to the problem could be of great help for plant biotechnologists. Results By using a quantitative real-time PCR method that requires limited preliminary optimisation steps, we achieved statistically significant estimates of 1, 2 and 3 copies of a transgene in the primary transformants. Furthermore, by estimating the copy number of both the gene of interest and the selectable marker gene, we show that rearrangements of the T-DNA are not the exception, and probably happen more often than usually recognised. Conclusions We have developed a rapid and reliable method to estimate the number of integrated copies following genetic transformation. Unlike other similar procedures, this method is not dependent on identical amplification efficiency between the PCR systems used and does not need preliminary information on a calibrator. Its flexibility makes it appropriate in those situations where an accurate optimisation of all reaction components is impossible or impractical. Finally, the quality of the information produced is higher than what can be obtained by Southern blot analysis.

  15. Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus

    Directory of Open Access Journals (Sweden)

    Kai Wang

    2017-04-01

    Full Text Available Quantitative real-time reverse transcription PCR (RT-qPCR has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different

  16. Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus.

    Science.gov (United States)

    Wang, Kai; Niu, Yi; Wang, Qijun; Liu, Haili; Jin, Yi; Zhang, Shenglin

    2017-01-01

    Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to

  17. Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

    Directory of Open Access Journals (Sweden)

    Tandale Babasaheb V

    2008-12-01

    Full Text Available Abstract Background Chandipura virus (CHPV, a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR using TaqMan technology was developed for rapid diagnosis. Methods Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice in ovo (eggs, in vitro (Vero E6, PS, RD and Sand fly cell line] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard. Results Real-time one step RT-PCR was optimized using in vitro transcribed (IVT RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99 with maximum Coefficient of variation (CV = 5.91% for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml. Vero and PS cell-lines (1.2 × 103 PFU/ml were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Science.gov (United States)

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

  20. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

    Science.gov (United States)

    Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster have been found to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. The United States Environmental Protection Agency is planning to conduct a ...

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  2. Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR

    NARCIS (Netherlands)

    Kuiper, M.W.; Valster, R.M.; Wullings, B.A.; Boonstra, H.; Smidt, H.; Kooij, van der D.

    2006-01-01

    A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic

  3. Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR

    NARCIS (Netherlands)

    Kuiper, M.W.; Valster, R.M.; Wullings, B.A.; Boonstra, H.; Smidt, H.; Kooij, van der D.

    2006-01-01

    A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic

  4. Validation and standardization of gene expression data for microarray and real time quantitative PCR using universal external RNA controls

    Science.gov (United States)

    This presentation will introduce newly developed universal external ribonucleic acid (RNA) controls and their applications on different platforms of microarray and quantitative real time polymerase chain reaction (qRT-PCR) including SYBR Green® and TaqMan® probe-based chemistries. Data obtained fro...

  5. Using Quantitative Real-Time PCR to Detect MicroRNA Expression Profile During Embryonic Stem Cell Differentiation.

    Science.gov (United States)

    Pan, Xiaoping; Murashov, Alexander K; Stellwag, Edmund J; Zhang, Baohong

    2017-01-01

    Quantitative real-time PCR (qRT-PCR) is a reliable method to determine and monitor microRNA (miRNA) expression profiles in different cells, tissues, and organisms. Although there are several different strategies in performing qRT-PCR to determine miRNA expression, all of them have two steps in common: reverse transcription for obtaining cDNA from mature miRNA sequencing and standard real-time PCR for amplification of cDNA. This chapter demonstrates the application of quantitative real-time PCR for determining miRNA expression profiles during mouse embryonic stem cell differentiation. In this method, a mature miRNA sequence is first reverse transcribed into a long cDNA with a 40-50 nt miRNA-specific stem-loop primer; then, a standard real-time PCR reaction is performed for determining miRNA expression using a forward miRNA-specific primer and a universal reverse primer.

  6. Simultaneous detection, typing and quantitation of oncogenic human papillomavirus by multiplex consensus real-time PCR.

    Science.gov (United States)

    Jenkins, Andrew; Allum, Anne-Gry; Strand, Linda; Aakre, Randi Kersten

    2013-02-01

    A consensus multiplex real-time PCR test (PT13-RT) for the oncogenic human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 is described. The test targets the L1 gene. Analytical sensitivity is between 4 and 400 GU (genomic units) in the presence of 500 ng of human DNA, corresponding to 75,000 human cells. HPV types are grouped into multiplex groups of 3 or 4 resulting in the use of 4 wells per sample and permitting up to 24 samples per run (including controls) in a standard 96-well real-time PCR instrument. False negative results are avoided by (a) measuring sample DNA concentration to control that sufficient cellular material is present and (b) including HPV type 6 as a homologous internal control in order to detect PCR inhibition or competition from other (non-oncogenic) HPV types. Analysis time from refrigerator to report is 8 h, including 2.5 h hands-on time. Relative to the HC2 test, the sensitivity and specificity were respectively 98% and 83%, the lower specificity being attributable to the higher analytical sensitivity of PT13-RT. To assess type determination comparison was made with a reversed line-blot test. Type concordance was high (κ=0.79) with discrepancies occurring mostly in multiple-positive samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time.

    Science.gov (United States)

    Sugita, Sunao; Ogawa, Manabu; Inoue, Shizu; Shimizu, Norio; Mochizuki, Manabu

    2011-09-01

    To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1 × 10(2)-2.1 × 10(6) copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

  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. 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.

  10. Evaluation of internal control for gene expression in Phalaenopsis by quantitative real-time PCR.

    Science.gov (United States)

    Yuan, Xiu-Yun; Jiang, Su-Hua; Wang, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2014-07-01

    The selection of appropriate reference genes is one of the most important steps to obtain reliable results for normalizing quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) of MADS-box gene in Phalaenopsis. In this study, we cloned 12 candidate reference genes including 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), cytoskeletal structural protein actin (ACT1, ACT2, ACT3, ACT4, ACT5), ubiquitin protein (UBQ1 and UBQ2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the cytoskeletal structural proteins α-tubulin (TUA) and β-tubulin (TUB) in Phalaenopsis and evaluated their expression reliability. The expression of these candidate reference genes was analyzed using geNorm and normFinder software packages; the results showed that ACT2 and ACT4 were the highest stability reference genes for all experiment sets based on normFinder, followed by ACT1 or ACT3, while ACT3 and ACT4 were the highest stability reference genes for most experiment sets based on geNorm, then TUB or others. Taken together, Actin genes were the higher stability reference genes for all tissues at total developmental stages, and similar results came from analysis by normFinder. According to geNorm analysis, ACT3 and ACT4 were the most stable reference genes for all tissues tested and tissues at reproductive stages; TUB and ACT5 or ACT4 were the most stable reference genes for vegetative tissues or roots. The most stable reference genes for all vegetative tissues and only leaves were ACT4 and ACT5, ACT2 and ACT3, respectively; ACT1 and ACT3 were the most stable genes and sufficient for reliable normalization of flower tissues. While EF1α, UBQ1, UBQ2, and GAPDH were found to be unsuitable as a reference gene in our analysis for flower tissues, total tissues, and reproductive stages; UBQ2 and 18S were identified as the least stable reference genes for vegetative tissues at different stages, different tissues at vegetative stages; TUA and 18S were the

  11. Effect of ionizing radiation on the quantitative detection of Salmonella using real-time PCR

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Sangyong; Jung, Jinwoo [Radiation Research Center for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Kim, Minjeong; Ryu, Sangryeol [Department of Food and Animal Biotechnology, School of Agricultural Biotechnology, Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921 (Korea, Republic of); Kim, Dongho [Radiation Research Center for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of)], E-mail: fungikim@kaeri.re.kr

    2008-09-15

    Food irradiation is an economically viable technology for inactivating foodborne pathogens, but irradiation can mask pathogens in unhygienically prepared food. The aim of this study was to investigate the effect of irradiation treatment on the detection of Salmonella using real-time PCR. Three commercially available kits were tested, of which the InstaGene Matrix procedure was most effective in preparing template DNA from Salmonella exposed to radiation in broth culture. The minimum level of detection by real-time PCR combined with InstaGene Matrix was 3 log units of Salmonella per milliliter. However, when pure cultures of Salmonella were irradiated at 3 and 5 kGy, the cycle threshold (C{sub T}) increased 1-1.5-fold compared to irradiation at 0 and 1 kGy. This indicated that irradiation treatment may result in an underestimation of bacterial counts due to radiation-induced DNA lesions. We also compared C{sub T} values in inoculated chicken homogenates before and after irradiation, which in this model caused a 1.3-3.3-fold underestimation of bacterial counts with respect to irradiation dose.

  12. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    Science.gov (United States)

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA.

  13. The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

    Directory of Open Access Journals (Sweden)

    A.C.M.F. Souza

    2009-09-01

    Full Text Available Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99% samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR, and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001. In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99% samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR, and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001. These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.

  14. Quantitative Analysis of Periodontal Pathogens Using Real-Time Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Marin, Mª José; Figuero, Elena; Herrera, David; Sanz, Mariano

    2017-01-01

    The quantitative polymerase chain reaction (qPCR) is a variant of PCR aimed to detect and quantify a targeted DNA molecule through the addition of probes labeled with fluorescent molecules that emit fluorescence within each amplification cycle, what results in fluorescence values proportional to the amount of accumulated PCR product. This chapter presents the detailed procedures for quantification of different periodontal pathogens (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, and Fusobacterium spp.) using qPCR. It also includes the description of the most frequent problems encountered and how to solve them. In addition, a detailed protocol for multiplex qPCR to detect and quantify P. gingivalis and A. actinomycetemcomitans is included.

  15. Sex ratio determination in bovine semen: a new approach by quantitative real time PCR.

    Science.gov (United States)

    Parati, K; Bongioni, G; Aleandri, R; Galli, A

    2006-12-01

    Sex preselection of livestock offspring in cattle represents, nowadays, a big potential for genetic improvement and market demand satisfaction. Sperm sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos but, an accurate verification of the yield of sperm separation remains essential for a field application of this technique or for improvement and validation of other related semen sexing technologies. In this work a new method for the determination of the proportion of X- and Y-bearing spermatozoa in bovine semen sample was developed by real time PCR. Two sets of primers and internal TaqMan probes were designed on specific X- and Y-chromosome genes. To allow a direct quantification, a standard reference was established using two plasmid cDNA clones (ratio 1:1) for the specific gene targets. The method was validated by a series of accuracy, repeatability and reproducibility assays and by testing two sets of sorted and unsorted semen samples. A high degree of accuracy (98.9%), repeatability (CV=2.58%) and reproducibility (CV=2.57%) was shown. The results of X- and Y-sorted semen samples analysed by real time PCR and by flow cytometric reanalysis showed no significant difference (P>0.05). The evaluation of X-chromosome bearing sperms content in unsorted samples showed an average of 51.11+/-0.56% for ejaculates and 50.17+/-0.58% for the commercial semen. This new method for quantification of the sexual chromosome content in spermatozoa demonstrated to be rapid and reliable, providing a valid support to the sperm sexing technologies.

  16. Simulation of collaborative studies for real-time PCR-based quantitation methods for genetically modified crops.

    Science.gov (United States)

    Watanabe, Satoshi; Sawada, Hiroshi; Naito, Shigehiro; Akiyama, Hiroshi; Teshima, Reiko; Furui, Satoshi; Kitta, Kazumi; Hino, Akihiro

    2013-01-01

    To study impacts of various random effects and parameters of collaborative studies on the precision of quantitation methods of genetically modified (GM) crops, we developed a set of random effects models for cycle time values of a standard curve-based relative real-time PCR that makes use of an endogenous gene sequence as the internal standard. The models and data from a published collaborative study for six GM lines at four concentration levels were used to simulate collaborative studies under various conditions. Results suggested that by reducing the numbers of well replications from three to two, and standard levels of endogenous sequence from five to three, the number of unknown samples analyzable on a 96-well PCR plate in routine analyses could be almost doubled, and still the acceptable repeatability RSD (RSDr crops by real-time PCR and their collaborative studies.

  17. Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

    Science.gov (United States)

    Bushon, R.N.; Kephart, C.M.; Koltun, G.F.; Francy, D.S.; Schaefer, F. W.; Lindquist, H.D. Alan

    2010-01-01

    Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

  18. Real-time quantitative PCR for detection and identification of Actinobacillus pleuropneumoniae serotype 2

    Directory of Open Access Journals (Sweden)

    Dors Arkadiusz

    2016-09-01

    Full Text Available Introduction: Porcine pleuropneumonia inflicts important economic losses on most commercial herds. Detection of subclinical or chronic infection in animals still remains a challenge, as isolation and identification of A. pleuropneumoniae serotypes is difficult and quantification of the bacteria on agar plates is often almost impossible. The aim of the study was to develop and evaluate a serotype-specific quantitative TaqMan probe-based PCR for detection of serotype 2 in pig lungs, tonsils, and nasal swabs.

  19. Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

    Science.gov (United States)

    Wong, Wilson; Farr, Ryan; Joglekar, Mugdha; Januszewski, Andrzej; Hardikar, Anandwardhan

    2015-01-01

    Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput. PMID:25938938

  20. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

    Science.gov (United States)

    Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

    2004-03-01

    Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

  1. 实时定量PCR技术及其应用%Real-time Quantitative PCR and Its Applications

    Institute of Scientific and Technical Information of China (English)

    王梁燕; 洪奇华; 张耀洲

    2004-01-01

    实时定量PCR(Real-time Quantitative Polymerase Chain Reaction,RQ-PCR)技术是20世纪90年代中期发展起来的一种新型核酸定量技术.该技术具有实时监测、快速、灵敏、精确等特点,是对原有PCR技术的革新,扩大了PCR的应用范围.本文综述了RQ-PCR技术的原理、RQ-PCR仪、RQ-PCR实时定量检测系统及其应用.

  2. 实时定量PCR技术及应用%Real-time quantitative PCR and its applications

    Institute of Scientific and Technical Information of China (English)

    杨凤秋; 朱正歌

    2006-01-01

    实时定量PCR(Real-time Quantitative Polymerase Chain Reaction,RQ-PCR),是20世纪90年代中期发展起来的基于PCR技术的利用不同的荧光检测来给核酸定量的技术.克服了传统PCR的许多不足,能准确敏感地检测模板浓度,DNA拷贝数和检测基因变异.综述了RQ-PCR技术的原理,RQ-PCR实时定量检测系统及应用.

  3. Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene.

    Science.gov (United States)

    Tobias, T J; Bouma, A; Klinkenberg, D; Daemen, A J J M; Stegeman, J A; Wagenaar, J A; Duim, B

    2012-08-01

    A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2010-03-01

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

  5. Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

    OpenAIRE

    Karatzas, Kimon Andreas G.

    2012-01-01

    Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count m...

  6. Molecular ABO phenotyping in cynomolgus macaques using real-time quantitative PCR.

    Science.gov (United States)

    Premasuthan, A; Ng, J; Kanthaswamy, S; Trask, J S; Houghton, P; Farkas, T; Sestak, K; Smith, D G

    2012-10-01

    Macaques are commonly used in biomedical research as animal models of human disease. The ABO phenotype of donors and recipients plays an important role in the success of transplantation and stem cell research of both human and macaque tissue. Traditional serological methods for ABO phenotyping can be time consuming, provide ambiguous results and/or require tissue that is unavailable or unsuitable. We developed a novel method to detect the A, B, and AB phenotypes of macaques using real-time quantitative polymerase chain reaction. This method enables the simple and rapid screening of these phenotypes in macaques without the need for fresh blood or saliva. This study reports the distribution of the A, B, and AB phenotypes of captive cynomolgus macaques that, while regionally variable, closely resembles that of rhesus macaques. Blood group B, as in rhesus macaques, predominates in cynomolgus macaques and its frequency distribution leads to a probability of major incompatibility of 41%. No silencing mutations have been identified in exon 6 or 7 in macaques that could be responsible for the O phenotype, that, although rare, have been reported. The excess homozygosity of rhesus and cynomolgus macaque genotypes in this study, that assumes the absence of the O allele, suggests the possibility of some mechanism preventing the expression of the A and B transferases.

  7. A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

    Directory of Open Access Journals (Sweden)

    Wilson Zoe A

    2008-06-01

    Full Text Available Abstract Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP, which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP and a complementary quenching probe (QP lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

  8. 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.

  9. Rapid quantitative detection of, Listeria monocytogenes in salmon products: evaluation of pre-real-time PCR strategies.

    Science.gov (United States)

    Rodríguez-Lázaro, David; Jofré, Anna; Aymerich, Teresa; Garriga, Margarita; Pla, Maria

    2005-07-01

    The spread and persistence of Listeria monocytogenes in smoked fish products and seafood processing factories are big concerns. Thus, the corresponding quality assurance programs must include adequate microbiological control measures. We evaluated eight different pre-PCR sample processing strategies to be coupled with a previously developed real-time PCR assay for the quantitative detection of L. monocytogenes in salmon products. The optimal pre-PCR procedure involved filtration and DNA purification with the use of a commercial kit. This strategy could detect 10 CFU of L. monocytogenes per g of smoked salmon and could quantify 1,000 CFU/g with excellent accuracy compared with the standard plate count method. Thus, this method could be a promising alternative for the quantitative detection of L. monocytogenes in smoked fish products and processing factories. This method could also detect the bacterium in raw salmon.

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

    Science.gov (United States)

    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

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

  11. Evaluation of reference genes for real-time quantitative PCR in the marine flavobacterium Zobellia galactanivorans.

    Science.gov (United States)

    Thomas, François; Barbeyron, Tristan; Michel, Gurvan

    2011-01-01

    The marine bacteria Zobellia galactanivorans is an emerging model microorganism for the bioconversion of algal polysaccharides. The sequence analysis of its genome opens the way to in-depth gene expression analysis, such as reverse transcription quantitative PCR (RT-qPCR) studies. The selection and validation of reference genes are a mandatory first step for the accurate quantification of transcripts. We selected fourteen candidate reference genes belonging to distinct pathways, namely replication, transcription, translation, citric acid cycle, amino acid, nucleotide and dihydrofolate metabolisms, and peptidoglycan, FMN and aromatic compounds synthesis. We quantified their expression by RT-qPCR in various culture conditions corresponding to different temperatures, carbon sources or stresses. The applications geNorm and Normfinder allowed ranking the genes according to their stability and gave concordant results. We found that the geometric average of the expression of glyA, icdA and gmkA can be confidently used to normalize the transcript abundance of genes of interest. In conclusion, this work provides a reliable procedure for gene expression analysis in the flavobacterium Z. galactanivorans and a validated set of reference genes to be used in future transcriptomics approaches. The strategy developed could also be the starting point for similar studies in other members of the Flavobacteria class.

  12. Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR.

    Science.gov (United States)

    Fykse, Else Marie; Olsen, Jaran Strand; Skogan, Gunnar

    2003-10-01

    A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.

  13. Quantitative analysis of colonization with real-time PCR to identify the role of Oxalobacter formigenes in calcium oxalate urolithiasis.

    Science.gov (United States)

    Batislam, Ertan; Yilmaz, Erdal; Yuvanc, Ercan; Kisa, Ozgul; Kisa, Ucler

    2012-10-01

    The objective of the study was to quantitatively measure the number of Oxalobacter formigenes (O. formigenes) colonizations in the gastrointestinal tract in calcium oxalate-forming patients with real-time polymerase chain reaction (PCR). Calcium oxalate-forming patients (n: 27) were included in the study. Serum calcium, sodium, potassium, urea and creatinine levels, as well as 24 h urine levels of calcium and oxalate were measured. The numbers of O. formigenes colonies in stool samples were detected by real-time PCR. One or two metabolic abnormalities were detected in 15 of 27 patients. The O. formigenes levels in patients with metabolic disturbance were significantly decreased when compared to the patients with no metabolic abnormalities (p: 0.038). The undetectable levels of O. formigenes were encountered in one of five patients with hypercalciuria, in three of four patients with hyperoxaluria and in four of six patients with both hypercalciuria and hyperoxaluria. In nine patients with a history of stone recurrence, O. formigenes colonization was significantly lower than the patients with the first stone attack (p: 0.001). O. formigenes formation ceased or significantly diminished in patients with calcium oxalate stones with a coexistence of both hyperoxaluria and hypercalciuria. The measurement of O. formigenes colonies by real-time PCR seemed to be an inconvenient and expensive method. For this reason, the real-time PCR measurements can be spared for the patients with stone recurrences and with metabolic abnormalities like hypercalciuria and hyperoxaluria. The exact measurement of O. formigenes may also help more accurate programming of O. formigenes-based treatments.

  14. Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

    Science.gov (United States)

    Takahashi, Teruyuki; Tamura, Masato; Asami, Yukihiro; Kitamura, Eiko; Saito, Kosuke; Suzuki, Tsukasa; Takahashi, Sachiko Nonaka; Matsumoto, Koichi; Sawada, Shigemasa; Yokoyama, Eise; Takasu, Toshiaki

    2008-05-01

    Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.

  15. Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

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    Jensen Jørgen

    2002-07-01

    Full Text Available Abstract Background Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods. Results We compared the performance of a newly developed real-time PCR with a conventional PCR method for detection of C. pneumoniae. The PCR methods were tested on reference samples containing C. pneumoniae DNA and on 136 nasopharyngeal samples from patients with a chronic cough. We found the same detection limit for the two methods and that clinical performance was equal for the real-time PCR and for the conventional PCR method, although only three samples tested positive. To investigate whether the low prevalence of C. pneumoniae among patients with a chronic cough was caused by suboptimal PCR efficiency in the samples, PCR efficiency was determined based on the real-time PCR. Seventeen of twenty randomly selected clinical samples had a similar PCR efficiency to samples containing pure genomic C. pneumoniae DNA. Conclusions These results indicate that the performance of real-time PCR is comparable to that of conventional PCR, but that needs to be confirmed further. Real-time PCR can be used to investigate the PCR efficiency which gives a rough estimate of how well the real-time PCR assay works in a specific sample type. Suboptimal PCR efficiency of PCR is not a likely explanation for the low positivity rate of C. pneumoniae in patients with a chronic cough.

  16. Modeling real-time PCR kinetics: Richards reparametrized equation for quantitative estimation of European hake (Merluccius merluccius).

    Science.gov (United States)

    Sánchez, Ana; Vázquez, José A; Quinteiro, Javier; Sotelo, Carmen G

    2013-04-10

    Real-time PCR is the most sensitive method for detection and precise quantification of specific DNA sequences, but it is not usually applied as a quantitative method in seafood. In general, benchmark techniques, mainly cycle threshold (Ct), are the routine method for quantitative estimations, but they are not the most precise approaches for a standard assay. In the present work, amplification data from European hake (Merluccius merluccius) DNA samples were accurately modeled by three sigmoid reparametrized equations, where the lag phase parameter (λc) from the Richards equation with four parameters was demonstrated to be the perfect substitute for Ct for PCR quantification. The concentrations of primers and probes were subsequently optimized by means of that selected kinetic parameter. Finally, the linear correlation among DNA concentration and λc was also confirmed.

  17. A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

    DEFF Research Database (Denmark)

    Kjær, L.; Riley, C.H.; Westman, M.

    2012-01-01

    present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel...... mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell...... populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B-lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay provides an important...

  18. Direct real-time quantitative PCR for measurement of host-cell residual DNA in therapeutic proteins.

    Science.gov (United States)

    Peper, Grit; Fankhauser, Alexander; Merlin, Thomas; Roscic, Ana; Hofmann, Matthias; Obrdlik, Petr

    2014-11-01

    Real-time quantitative PCR (qPCR) is important for quantification of residual host cell DNA (resDNA) in therapeutic protein preparations. Typical qPCR protocols involve DNA extraction steps complicating sample handling. Here, we describe a "direct qPCR" approach without DNA extraction. To avoid interferences of DNA polymerase with a therapeutic protein, proteins in the samples were digested with proteinase K (PK) in the presence of sodium dodecyl sulfate (SDS). Tween 20 and NaCl were included to minimize precipitation of therapeutic proteins in the PK/SDS mix. After PK treatment, the solution was applied directly for qPCR. Inhibition of DNA polymerase by SDS was prevented by adding 2% (v/v) of Tween 20 to the final qPCR mix. The direct qPCR approach was evaluated for quantification of resDNA in therapeutic proteins manufactured in Chinese hamster ovary (CHO) host cells. First, direct qPCR was compared with qPCR applied on purified DNA ("extraction qPCR"). For both qPCRs, the same CHO-specific primers and probes were used. Comparable residual DNA levels were detected with both PCR approaches in purified and highly concentrated drug proteins as well as in in-process-control samples. Finally, the CHO-specific direct qPCR protocol was validated according to ICH guidelines and applied for 25 different therapeutic proteins. The specific limits of quantification were 0.1-0.8ppb for 24 proteins, and 2.0ppb for one protein. General applicability of the direct qPCR was demonstrated by applying the sample preparation protocol for quantification of resDNA in therapeutic proteins manufactured in other hosts such as Escherichia coli and mouse cells.

  19. Evaluation of methods for oligonucleotide array data via quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Morris Daryl E

    2006-01-01

    Full Text Available Abstract Background There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR is generally considered the "gold-standard" assay for measuring gene expression by biologists and is often used to confirm findings from microarray data. Here we use qRT-PCR measurements to validate methods for the components of processing oligo array data: background adjustment, normalization, mismatch adjustment, and probeset summary. An advantage of our approach over spike-in studies is that methods are validated on a real dataset that was collected to address a scientific question. Results We initially identify three of six popular methods that consistently produced the best agreement between oligo array and RT-PCR data for medium- and high-intensity genes. The three methods are generally known as MAS5, gcRMA, and the dChip mismatch mode. For medium- and high-intensity genes, we identified use of data from mismatch probes (as in MAS5 and dChip mismatch and a sequence-based method of background adjustment (as in gcRMA as the most important factors in methods' performances. However, we found poor reliability for methods

  20. Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR.

    Science.gov (United States)

    Kuiper, Melanie W; Valster, Rinske M; Wullings, Bart A; Boonstra, Harry; Smidt, Hauke; van der Kooij, Dick

    2006-09-01

    A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 x 10(-1) and 1.14 x 10(4) cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 +/- 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (> or =98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.

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

    Science.gov (United States)

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

    2016-09-01

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

  2. A Quantitative Real-Time PCR Approach for Assessing Campylobacter jejuni and Campylobacter coli Colonization in Broiler Herds.

    Science.gov (United States)

    Haas, Katrin; Overesch, Gudrun; Kuhnert, Peter

    2017-04-01

    Human campylobacteriosis is a major public health concern in developed countries, with Campylobacter jejuni and Campylobacter coli from poultry recognized as the main source of human infection. Identification of Campylobacter-positive broiler herds before slaughter is essential for implementing measures to avoid carryover of pathogens via the slaughter process into the food chain. However, appropriate methods that have been validated for testing poultry flocks antemortem are lacking for Campylobacter. A quantitative real-time PCR (qPCR) that allows simultaneous detection and quantification of C. jejuni and C. coli was adapted and optimized to be applied on boot socks. The adjusted qPCR serves as an easy, sensitive, and quantitative method for Campylobacter detection in poultry flocks antemortem by analysis of boot socks. An adequate correlation was found between qPCR and culture, as well as between boot socks and cecal samples, which are regarded as the "gold standard." Therefore, boot sock sampling followed by qPCR analysis provides a reliable and simple method for assessing Campylobacter load within a flock prior to slaughter. The approach allows categorization of broiler herds into negative, low, moderate, or high Campylobacter colonization. Based on the results of this new approach, risk assessment models, such as evaluating the possible effect of sorting flocks before slaughter, can be easily implemented. Similarly, targeted identification of highly colonized flocks for improvement of biosecurity measures at the farm level will become feasible, presenting an opportunity to increase food safety.

  3. Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    Science.gov (United States)

    Takabatake, Reona; Koiwa, Tomohiro; Kasahara, Masaki; Takashima, Kaori; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Oguchi, Taichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

    2011-01-01

    To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

  4. A Quantitative Real-Time PCR-Based Strategy for Molecular Evaluation of Nicotine Conversion in Burley Tobacco

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    Bo Sun

    2015-11-01

    Full Text Available Nornicotine production in Nicotiana tabacum is undesirable because it is the precursor of the carcinogen N′-nitrosonornicotine. In some individual burley tobacco plants, a large proportion of the nicotine can be converted to nornicotine, and this process of nicotine conversion is mediated primarily by enzymatic N-demethylation of nicotine which is controlled mainly by CYP82E4. Here we report a novel strategy based on quantitative real-time polymerase chain reaction (qPCR method, which analyzed the ratio of nicotine conversion through examining the transcript level of CYP82E4 in burley leaves and do not need ethylene induction before detected. The assay was linear in a range from 1 × 101 to 1 × 105 copies/mL of serially diluted standards, and also showed high specificity and reproducibility (93%–99%. To assess its applicability, 55 plants of burley cultivar Ky8959 at leaf maturing stage were analyzed, and the results were in accordance with those from gas chromatograph-mass spectrometry (GC-MS method. Moreover, a linear correlation existed between conversion level and CYP82E4 transcript abundance. Taken together, the quantitative real-time PCR assay is standardized, rapid and reproducible for estimation of nicotine conversion level in vivo, which is expected to shed new light on monitoring of burley tobacco converter.

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

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    Borges-Pérez Andrés

    2008-12-01

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

  6. Validation and application of a PCR primer set to quantify fungal communities in the soil environment by real-time quantitative PCR.

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    Nicolas Chemidlin Prévost-Bouré

    Full Text Available Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR. The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1/FF390. This in silico analysis of the specificity of FR1/FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1/FF390 for Fungi was validated in vitro by cloning--sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils.

  7. Progress in real-time quantitative PCR technique%实时荧光定量PCR技术进展

    Institute of Scientific and Technical Information of China (English)

    洪云; 李津; 汪和睦; 赵铠

    2006-01-01

    实时荧光定量PCR(real-time quantitative PCR)技术是一种新型的核酸定量检测、分析技术,它通过在PCR扩增反应过程中加入荧光物质,使得对反应过程的实时监控成为可能.它具有实时监测、定量准确、灵敏度高、反应速度快、重复性好及PCR反应后不需电泳检测等优点,已逐步成为分子生物学研究中的重要工具.

  8. A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples.

    Science.gov (United States)

    Behets, Jonas; Declerck, Priscilla; Delaedt, Yasmine; Verelst, Lieve; Ollevier, Frans

    2007-01-01

    A fast and accurate duplex real-time PCR (qPCR) was developed to detect and quantify the human pathogenic amoeba Naegleria fowleri in water samples. In this study, primers and probe based on the Mp2Cl5 gene were designed to amplify and quantify N. fowleri DNA in a single duplex reaction. The qPCR detection limit (DL) corresponds to the minimum DNA quantity showing significant fluorescence with at least 90% of the positive controls in a duplex reaction. Using fluorescent Taqman technology the qPCR was found to be 100% specific for N. fowleri with a DL of 3 N. fowleri cell equivalents and a PCR efficiency of 99%. The quantification limit (QL) was 16 N. fowleri cell equivalents (corresponded with 320 N. fowleri cell equivalents l(-1) water sample) in a duplex qPCR reaction and corresponds to the lowest DNA quantity amplifiable with a coefficient of variation less than 25%. To detect inhibition an exogenous internal positive control (IPC) was included in each PCR reaction preventing false negative results. Comparison of qPCR and most probable number (MPN) culture results confirms that the developed qPCR is well suited for rapid and quantitative detection of this human pathogen in real water samples. Nevertheless 'low contamination levels' of water samples (fowleri cells l(-1)) still require culture method analyses. When other thermophilic Naegleria are very dominant, the MPN culture method could result in an underestimation in the real number of N. fowleri and some caution is necessary to interpret the data. The N. fowleri qPCR could be a useful tool to study further competitive phenomena between thermophilic Naegleria strains.

  9. O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

    Science.gov (United States)

    Mekonnen, Solomon A; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz

    2017-10-02

    Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

  10. Performance Assessment of Human and Cattle Associated Quantitative Real-time PCR Assays - slides

    Science.gov (United States)

    The presentation overview is (1) Single laboratory performance assessment of human- and cattle associated PCR assays and (2) A Field Study: Evaluation of two human fecal waste management practices in Ohio watershed.

  11. Comparison of real-time quantitative PCR and culture for the diagnosis of emerging Rickettsioses.

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    Emmanouil Angelakis

    Full Text Available BACKGROUND: Isolation of Rickettsia species from skin biopsies may be replaced by PCR. We evaluated culture sensitivity compared to PCR based on sampling delay and previous antibiotic treatment. METHODOLOGY/PRINCIPAL FINDINGS: Skin biopsies and ticks from patients with suspected Rickettsia infection were screened for Rickettsia spp. using qPCR, and positive results were amplified and sequenced for the gltA and ompA genes. Immunofluorescence for spotted fever group rickettsial antigens was done for 79 patients. All skin biopsies and only ticks that tested positive using qPCR were cultured in human embryonic lung (HEL fibroblasts using the centrifugation-shell vial technique. Patients and ticks were classified as definitely having rickettsioses if there was direct evidence of infection with a Rickettsia sp. using culture or molecular assays or in patients if serology was positive. Data on previous antibiotic treatments were obtained for patients with rickettsiosis. Rickettsia spp. infection was diagnosed in 47 out of 145 patients (32%, 41 by PCR and 12 by culture, whereas 3 isolates were obtained from PCR negative biopsies. For 3 of the patients serology was positive although PCR and culture were negative. Rickettsia africae was the most common detected species (n = 25, [17.2%] and isolated bacterium (n = 5, [3.4%]. The probability of isolating Rickettsia spp. was 12 times higher in untreated patients and 5.4 times higher in patients from our hometown. Rickettsia spp. was amplified in 24 out of 95 ticks (25% and we isolated 7 R. slovaca and 1 R. raoultii from Dermacentor marginatus. CONCLUSIONS/SIGNIFICANCE: We found a positive correlation between the bacteria copies and the isolation success in skin biopsies and ticks. Culture remains critical for strain analysis but is less sensitive than serology and PCR for the diagnosis of a Rickettsia infection.

  12. Detection and quantitation of the new world Squash leaf curl virus by TaqMan real-time PCR.

    Science.gov (United States)

    Abrahamian, Peter E; Abou-Jawdah, Yusuf

    2013-07-01

    Squash leaf curl diseases are caused by distinct virus species that are separated into two major phylogenetic groups, western and eastern hemisphere groups. The western group includes the new world Squash leaf curl virus (SLCV) which causes major losses to cucurbit production and induces severe stunting and leaf curl in squash plants. A TaqMan-based real time polymerase chain reaction (qPCR) assay has been developed for detection and quantitation of SLCV. Designed primers and probe targeted the AV1 (coat protein) gene and in silico analysis showed that they detect a large number of SLCV isolates. The developed assay could detect the virus in 18fg of total nucleic acid and 30 genomic units. The qPCR assay was about 1000 times more sensitive than PCR and amplified successfully SLCV from a wide range of cucurbit hosts and from viruliferous whiteflies. The developed qPCR assay should be suitable for detection and quantitation purposes for all reported SLCV isolates of the western hemisphere.

  13. SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case

    Directory of Open Access Journals (Sweden)

    Costa Elena

    2011-05-01

    Full Text Available Abstract Background 22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series of patients with congenital heart defects. A technical issue related to the VPREB1 qPCR marker was pointed out. Methods A set of 100 unrelated Italian patients with congenital heart defects were tested for 22q11.2 microdeletions by a qPCR method using six different markers. Fluorescence In Situ Hybridization technique (FISH was used for confirmation. Results qPCR identified six patients harbouring the 22q11.2 microdeletion, confirmed by FISH. The VPREB1 gene marker presented with a pattern consistent with hemideletion in one 3 Mb deleted patient, suggestive for a long distal deletion, and in additional five non-deleted patients. The long distal 22q11.2 deletion was not confirmed by Comparative Genomic Hybridization. Indeed, the VPREB1 gene marker generated false positive results in association with the rs1320 G/A SNP, a polymorphism localized within the VPREB1 marker reverse primer sequence. Patients heterozygous for rs1320 SNP, showed a qPCR profile consistent with the presence of a hemideletion. Conclusions Though the qPCR technique showed advantages as a screening approach in terms of cost and time, the VPREB1 marker case revealed that single nucleotide polymorphisms can interfere with qPCR data generating erroneous allelic copy number interpretations.

  14. Quantitative real-time PCR of enteric viruses in influent and effluent samples from wastewater treatment plants in Italy.

    Science.gov (United States)

    La Rosa, Giuseppina; Pourshaban, Manoochehr; Iaconelli, Marcello; Muscillo, Michele

    2010-01-01

    The prevalence of enteric viruses in wastewater, the efficacy of wastewater treatments in eliminating such viruses, and potential health risks from their release into the environment or by recycling of treated wastewaters, are very important issues in environmental microbiology. In this study we performed a quantitative TaqMan real-time PCR (polymerase chain reaction) analysis of enteric viruses on samples of influents and effluents from 5 wastewater treatment plants in and around Rome. Three epidemiologically important, waterborne enteric viruses were analyzed: adenoviruses, enteroviruses and noroviruses (GI and GII) and compared to classical bacterial indicators of fecal contamination. The concentration of adenoviruses was the highest, in both raw and treated waters. Mean values in influents were ranked as follows: adenovirus > norovirus GI > norovirus GII > enterovirus. In effluents, the ranking was: adenovirus > norovirus GI > enterovirus > norovirus GII. Removal efficiencies ranged from 35% (enterovirus) to 78% (norovirus GI), while removal efficiency for bacterial indicators was up to 99%. Since molecular quantification does not necessarily indicate an actual threat to human health, we proceeded to evaluate the infectivity of enterovirus particles in treated effluents through integrated cell culture and real-time PCR. Infectivity assays detected live virions in treated water, pointing to potential public health risks through the release of these viruses into the environment. A better understanding of viral presence and resistance to sewage purification processes have the potential of contributing to the effective management of risks linked to the recycling of treated wastewater, and its discharge into the environment.

  15. Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay.

    Science.gov (United States)

    Kao, Po-Min; Tung, Min-Che; Hsu, Bing-Mu; Chiu, Yi-Chou; She, Cheng-Yu; Shen, Shu-Min; Huang, Yu-Li; Huang, Wen-Chien

    2013-09-01

    In this study, a SYBR green quantitative real-time PCR was developed to quantify and detect the Legionella spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and thermal spring area in Taiwan. Legionella was detected in 13.6 % (24/176), and the detection rate for river water, raw drinking water, and thermal spring water was 10, 21.4, and 16.6 %, respectively. Using real-time PCR, concentration of Legionella spp. in detected samples ranged between 9.75 × 10(4) and 3.47 × 10(5) cells/L in river water, 6.92 × 10(4) and 4.29 × 10(5) cells/L in raw drinking water, and 5.71 × 10(4) and 2.12 × 10(6) cells/L for thermal spring water samples. The identified species included Legionella pneumophila (20.8 %), Legionella jordanis (4.2 %), Legionella nautarum (4.2 %), Legionella sp. (4.2 %), and uncultured Legionella sp. (66.6 %). The presence of L. pneumophila in aquatic environments suggested a potential public health threat that must be further examined.

  16. Accurate and objective copy number profiling using real-time quantitative PCR.

    Science.gov (United States)

    D'haene, Barbara; Vandesompele, Jo; Hellemans, Jan

    2010-04-01

    Copy number changes are known to be involved in numerous human genetic disorders. In this context, qPCR-based copy number screening may serve as the method of choice for targeted screening of the relevant disease genes and their surrounding regulatory landscapes. qPCR has many advantages over alternative methods, such as its low consumable and instrumentation costs, fast turnaround and assay development time, high sensitivity and open format (independent of a single supplier). In this chapter we provide all relevant information for a successfully implement of qPCR-based copy number analysis. We emphasize the significance of thorough in silico and empirical validation of the primers, the need for a well thought-out experiment design, and the importance of quality controls along the entire workflow. Furthermore, we suggest an appropriate and practical way to calculate copy numbers and to objectively interpret the results. The provided guidelines will most certainly improve the quality and reliability of your qPCR-based copy number screening.

  17. A highly sensitive quantitative real-time PCR assay for determination of mutant JAK2 exon 12 allele burden.

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    Lasse Kjær

    Full Text Available Mutations in the Janus kinase 2 (JAK2 gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B-lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.

  18. Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue.

    Science.gov (United States)

    Zhang, Wan-Xia; Fan, Jie; Ma, Jing; Rao, Yi-Song; Zhang, Li; Yan, You-E

    2016-06-22

    Quantitative real-time PCR (qRT-PCR) is the most classical technique in the field of gene expression study. This method requires an appropriate reference gene to normalize mRNA levels. In this study, the expression stability of four frequently-used reference genes in epididymal white adipose tissue (eWAT), inguinal beige adipose tissue (iBeAT) and brown adipose tissue (BAT) from obese and lean rats were evaluated by geNorm, NormFinder and BestKeeper. Based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, the two most stable reference genes were recommended in each type of adipose tissue. Two target genes were applied to test the stability of the reference genes. The geNorm and NormFinder results revealed that GAPDH and 36B4 exhibited the highest expression stabilities in eWAT, while 36B4 and β-actin had the highest expression stabilities in iBeAT and BAT. According to the results of the BestKeeper analysis, 36B4 was the most stable gene in eWAT, iBeAT and BAT, in terms of the coefficient of variance. In terms of the coefficient of correlation, GAPDH, 36B4 and β-actin were the most stable genes in eWAT, iBeAT and BAT, respectively. Additionally, expected results and statistical significance were obtained using a combination of two suitable reference genes for data normalization. In conclusion, 36B4 and GAPDH, in combination, are the best reference genes for eWAT, while 36B4 and β-actin are two most suitable reference genes for both iBeAT and BAT. We recommend using these reference genes accordingly.

  19. Comparative validation using quantitative real-time PCR (qPCR and conventional PCR of bovine semen centrifuged in continuous density gradient

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    M.V. Resende

    2011-06-01

    Full Text Available The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05. The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

  20. Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

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    Štrukelj Borut

    2008-03-01

    Full Text Available Abstract Background Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed. Results To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95°C for 10 minutes prior to storage at -20°C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25°C and the correlation between PCN and protein accumulation was established. Conclusion Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.

  1. Leptin receptor (LEPR SNP polymorphisms in HELLP syndrome patients determined by quantitative real-time PCR and melting curve analysis

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    Than Nándor

    2010-02-01

    Full Text Available Abstract Background Several studies have shown overexpression of leptin in microarray experiments in pre-eclampsia (PE and in hemolysis, elevated liver enzymes, low platelets (HELLP syndrome. We decided to study four leptin receptor (LEPR SNP polymorphisms in HELLP syndrome patients by using quantitative real-time PCR and melting curve analysis. Methods DNA was isolated from blood samples from 83 normotensive pregnant women and 75 HELLP syndrome patients. Four SNPs, LEPR c.326A>G (K109, LEPR c.668A>G (Q223R, LEPR c.1968G>C (K656N and LEPR c.3024A>G (S1008 were determined by quantitative real-time PCR and melting curve analysis. Investigators were blinded to clinical outcomes. Results LEPR c.326A>G, LEPR c.668A>G, LEPR c.1968G>C and LEPR c.3024A>G allele, genotype and haplotype polymorphisms were not different in HELLP syndrome patients and normotensive healthy pregnants. There were strong linkage disequilibrium (LD between loci c.326A>G and c.6687A>G (D' = 0.974, and c.668A>G and c.1968G>C (D' = 0.934, and c.326A>G and c.1968G>C (D' = 0.885, and c.1968G>C and c.3024A>G (D' = 1.0. However, linkages of c.3024A>G with c.668A>G (D' = 0.111 and c.326A>G (D' = 0.398 were weak. The Hardy-Weinberg equilibrium was observed for all polymorphisms. However the LEPR c.326A>G AG genotype was twice more frequent and the (AG AG GG AG haplotype was three times more frequent in HELLP syndrome patients. The introduced quantitative real-time PCR combined with melting curve analysis is a fast and reliable method for the determination of LEPR SNPs. Conclusion Although certain LEPR haplotypes are more frequent in HELLP syndrome, we conclude that there is no compelling evidence that the four studied LEPR SNP polymorphisms associated with the development of HELLP syndrome.

  2. 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.

  3. A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

    Science.gov (United States)

    Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

    2014-01-01

    Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Quantitative real time PCR detection of Clostridium difficile growth inhibition by probiotic organisms

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    Bryan L Folkers

    2010-01-01

    Full Text Available Background : Probiotic microorganisms are potential treatments for Clostridium difficile diarrheal disease (CDD but better methods are needed to determine the relative potency of probiotic microorganisms against pathogenic organisms in mixed cultures. Aim: Quantify C. difficile in the presence of putative probiotic organisms using molecular methods to determine relative probiotic potency. Materials and Methods: C. difficile strains were cultivated anaerobically. Serial dilutions of Lactobacillus cultures or microbial mixtures from kefir were co-cultured with C. difficile for 48 hours. Bacterial DNA was extracted and qPCR was used to measure C. difficile toxin A gene, on the basis of cycle threshold (Ct number. Results: Strains of Lactobacillus (human and ATCC derived, and mixed cultures from commercial kefir were co-cultured with C. difficile. Lactobacillus and the microbial mixture from kefir were ranked in order of their potency in C. difficile growth inhibition. Conclusions: PCR allows facile quantification of C. difficle in the presence of other. The technique measures relative potency of over-the-counter probiotics and may predict human strains meriting probiotic status.

  5. Quantitative real time PCR detection of Clostridium difficile growth inhibition by probiotic organisms

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    Michael Essmann

    2010-01-01

    Full Text Available Background: Probiotic microorganisms are potential treatments for Clostridium difficile diarrheal disease (CDD but better methods are needed to determine the relative potency of probiotic microorganisms against pathogenic organisms in mixed cultures. Aim: Quantify C. difficile in the presence of putative probiotic organisms using molecular methods to determine relative probiotic potency. Materials and Methods: C. difficile strains were cultivated anaerobically. Serial dilutions of Lactobacillus cultures or microbial mixtures from kefir were co-cultured with C. difficile for 48 hours. Bacterial DNA was extracted and qPCR was used to measure C. difficile toxin A gene, on the basis of cycle threshold (Ct number. Results: Strains of Lactobacillus (human and ATCC derived, and mixed cultures from commercial kefir were co-cultured with C. difficile. Lactobacillus and the microbial mixture from kefir were ranked in order of their potency in C. difficile growth inhibition. Conclusions: PCR allows facile quantification of C. difficle in the presence of other. The technique measures relative potency of over-the-counter probiotics and may predict human strains meriting probiotic status.

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

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    Pengfei Lin

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

  7. A quantitative real-time PCR method using an X-linked gene for sex typing in pigs.

    Science.gov (United States)

    Ballester, Maria; Castelló, Anna; Ramayo-Caldas, Yuliaxis; Folch, Josep M

    2013-06-01

    At present, a wide range of molecular sex-typing protocols in wild and domestic animals are available. In pigs, most of these methods are based on PCR amplification of X-Y homologous genes followed by gel electrophoresis which is time-consuming and in some cases expensive. In this paper, we describe, for the first time, a SYBR green-based quantitative real-time PCR (qPCR) assay using an X-linked gene, the glycoprotein M6B, for genetic sexing of pigs. Taking into account the differences in the glycoprotein M6B gene copy number between genders, we determine the correct sex of 54 pig samples from either diaphragm or hair follicle from different breeds using the 2(-ΔΔCT) method for relative quantification. Our qPCR assay represents a quick, inexpensive, and reliable tool for sex determination in pigs. This new protocol could be easily adapted to other species in which the sex determination was required.

  8. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti.

    Science.gov (United States)

    Dzaki, Najat; Ramli, Karima N; Azlan, Azali; Ishak, Intan H; Azzam, Ghows

    2017-03-16

    The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti, a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin (ACT), eukaryotic elongation factor 1 alpha (eEF1α), alpha tubulin (α-tubulin), ribosomal proteins L8, L32 and S17 (RPL8, RPL32 and RPS17), and glyceraldeyde 3-phosphate dehydrogenase (GAPDH) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research.

  9. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti

    Science.gov (United States)

    Dzaki, Najat; Ramli, Karima N.; Azlan, Azali; Ishak, Intan H.; Azzam, Ghows

    2017-01-01

    The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti, a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin (ACT), eukaryotic elongation factor 1 alpha (eEF1α), alpha tubulin (α-tubulin), ribosomal proteins L8, L32 and S17 (RPL8, RPL32 and RPS17), and glyceraldeyde 3-phosphate dehydrogenase (GAPDH) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research. PMID:28300076

  10. Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

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    Pricila da Silva Cunha

    2014-01-01

    Full Text Available Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH, which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH, and/or multiplex ligation-dependent probe amplification (MLPA all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

  11. Successful Validation of Sample Processing and Quantitative Real-Time PCR Capabilities on the International Space Station

    Science.gov (United States)

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

    2016-01-01

    The WetLab-2 system was developed by NASA Ames Research Center to offer new capabilities to researchers. The system can lyse cells and extract RNA (Ribonucleic Acid) on-orbit from different sample types ranging from microbial cultures to animal tissues. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (Polymerase Chain Reaction) (qRT-PCR) analysis without the need for sample return. The qRT-PCR results can be downlinked to the ground a few hours after the completion of the run. The validation flight of the WetLab-2 system launched on SpaceX-8 on April 8, 2016. On orbit operations started on April 15th with system setup and was followed by three quantitative PCR runs using an E. coli genomic DNA template pre-loaded at three different concentrations. These runs were designed to discern if quantitative PCR functions correctly in microgravity and if the data is comparable to that from the ground control runs. The flight data showed no significant differences compared to the ground data though there was more variability in the values, this was likely due to the numerous small bubbles observed. The capability of the system to process samples and purify RNA was then validated using frozen samples prepared on the ground. The flight data for both E. coli and mouse liver clearly shows that RNA was successfully purified by our system. The E. coli qRT-PCR run showed successful singleplex, duplex and triplex capability. Data showed high variability in the resulting Cts (Cycle Thresholds [for the PCR]) likely due to bubble formation and insufficient mixing during the procedure run. The mouse liver qRT-PCR run had successful singleplex and duplex reactions and the variability was slightly better as the mixing operation was improved. The ability to purify and stabilize RNA and to conduct qRT-PCR on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. The

  12. Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp. bulgaricus.

    Science.gov (United States)

    Shao, Yuyu; Wang, Zhaoxia; Bao, Qiuhua; Zhang, Heping

    2016-12-01

    In this study, a combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) was used to develop a method to determine the viability of cells of Lactobacillus delbrueckii ssp. bulgaricus ND02 (L. bulgaricus) that may have entered into a viable but nonculturable state. This can happen due to its susceptibility to cold shock during lyophilization and storage. Propidium monoazide concentration, PMA incubation time, and light exposure time were optimized to fully exploit the PMA-qPCR approach to accurately assess the total number of living L. bulgaricus ND02. Although PMA has little influence on living cells, when concentrations of PMA were higher than 30μg/mL the number of PCR-positive living bacteria decreased from 10(6) to 10(5) cfu/mL in comparison with qPCR enumeration. Mixtures of living and dead cells were used as method verification samples for enumeration by PMA-qPCR, demonstrating that this method was feasible and effective for distinguishing living cells of L. bulgaricus when mixed with a known number of dead cells. We suggest that several conditions need to be studied further before PMA-qPCR methods can be accurately used to distinguish living from dead cells for enumeration under more realistic sampling situations. However, this research provides a rapid way to enumerate living cells of L. bulgaricus and could be used to optimize selection of cryoprotectants in the lyophilization process and develop technologies for high cell density cultivation and optimal freeze-drying processes.

  13. Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater.

    Science.gov (United States)

    Varma, M; Field, R; Stinson, M; Rukovets, B; Wymer, L; Haugland, R

    2009-11-01

    A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. These methods were used in the analyses of wastewater samples to investigate their feasibility as alternatives to current fecal indicator bacteria culture methods for predicting the efficiency of viral pathogen removal by standard treatment processes. PMA treatment was effective in preventing qPCR detection of target sequences from non-viable cells. Concentrates of small volume, secondary-treated wastewater samples, collected from a publicly owned treatment works (POTW) under normal operating conditions, had little influence on this effectiveness. Higher levels of total suspended solids, such as those associated with normal primary treatment and all treatment stages during storm flow events, appeared to interfere with PMA effectiveness under the sample preparation conditions employed. During normal operating conditions at three different POTWs, greater reductions were observed in PMA-qPCR detectable target sequences of both Enterococcus and Bacteroidales than in total qPCR detectable sequences. These reductions were not as great as those observed for cultivable fecal indicator bacteria in response to wastewater disinfection. Reductions of PMA-qPCR as well as total qPCR detectable target sequences from enterococci and, to a lesser extent, Bacteroidales correlated well with reductions in infectious viruses during both normal and storm flow operating conditions and therefore may have predictive value in determining the efficiency at which these pathogens are removed.

  14. Detection of Anaplasma platys in dogs and Rhipicephalus sanguineus group ticks by a quantitative real-time PCR.

    Science.gov (United States)

    Ramos, Rafael Antonio Nascimento; Latrofa, Maria Stefania; Giannelli, Alessio; Lacasella, Vita; Campbell, Bronwyn Evelyn; Dantas-Torres, Filipe; Otranto, Domenico

    2014-09-15

    Anaplasma platys is an obligate intracellular, tick-borne pathogen of dogs, which causes canine infectious cyclic thrombocytopenia (CICT). The vector role of Rhipicephalus sanguineus group ticks has been only suggested, but definitive evidence is lacking. This study aimed to detect and quantify A. platys DNA in infected dogs and in their respective ticks through a quantitative real-time PCR assay. From March to May 2009, blood and tick samples from dogs residing in a CICT-endemic area were collected and molecularly analysed. Differences (p0.05). The mean bacterial load detected in positive dogs was lower than that in their respective ticks (p>0.05). This study provides circumstantial evidence of the putative role of Rhipicephalus sp. I as a vector of this pathogen. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Quantitative Real Time PCR approach to study gene expression profile during prenatal growth of skeletal muscle in pig of Duroc and Pietrain breeds

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    M. Cagnazzo

    2010-01-01

    Full Text Available The quantitative real time-PCR (QRT-PCR is a very sensitive method used to quantify mRNA level in gene expression analysis. Combining amplification, detection and quantification in a single step, allows a more accurate measurement compared to the traditional PCR end point analysis (Pfaffl, 2001; Bustin, 2002.

  16. Quantitative detection of Borrelia burgdorferi sensu lato in erythema migrans skin lesions using internally controlled duplex real time PCR.

    Science.gov (United States)

    O'Rourke, Maria; Traweger, Andreas; Lusa, Lara; Stupica, Dasa; Maraspin, Vera; Barrett, P Noel; Strle, Franc; Livey, Ian

    2013-01-01

    B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. bavariensis are the principal species which account for Lyme borreliosis (LB) globally. We have developed an internally controlled duplex quantitative real time PCR assay targeting the Borrelia 16S rRNA and the human RNAseP genes. This assay is well-suited for laboratory confirmation of suspected cases of LB and will be used to assess the efficacy of a vaccine against LB in clinical trials. The assay is highly specific, successfully detecting DNA extracted from 83 diverse B. burgdorferi sensu lato strains representing all major species causing LB, while 21 unrelated microbial species and human genomic DNA tested negative. The assay was highly reproducible and sensitive, with a lower limit of detection of 6 copies per PCR reaction. Together with culture, the assay was used to evaluate paired 3 mm skin biopsy samples taken from 121 patients presenting with solitary erythema migrans (EM) lesion. PCR testing identified more positive biopsy samples than culture (77.7% PCR positive versus 55.1% culture positive) and correctly identified all specimens scored as culture positive. OspA-based typing identified the majority of isolates as B. afzelii (96.8%) and the bacterial load was significantly higher in culture positive biopsies than in culture negative biopsies (Phistory of LB (P = 0.10). This is the first quantitative PCR study of human skin biopsies predominantly infected with B. afzelii and the first study to demonstrate a clear relationship between clinical symptoms in B. afzelii-infected patients and Borrelia burden.

  17. Standardisation of data from real-time quantitative PCR methods – evaluation of outliers and comparison of calibration curves

    Directory of Open Access Journals (Sweden)

    Burns Malcolm J

    2005-12-01

    Full Text Available Abstract Background As real-time quantitative PCR (RT-QPCR is increasingly being relied upon for the enforcement of legislation and regulations dependent upon the trace detection of DNA, focus has increased on the quality issues related to the technique. Recent work has focused on the identification of factors that contribute towards significant measurement uncertainty in the real-time quantitative PCR technique, through investigation of the experimental design and operating procedure. However, measurement uncertainty contributions made during the data analysis procedure have not been studied in detail. This paper presents two additional approaches for standardising data analysis through the novel application of statistical methods to RT-QPCR, in order to minimise potential uncertainty in results. Results Experimental data was generated in order to develop the two aspects of data handling and analysis that can contribute towards measurement uncertainty in results. This paper describes preliminary aspects in standardising data through the application of statistical techniques to the area of RT-QPCR. The first aspect concerns the statistical identification and subsequent handling of outlying values arising from RT-QPCR, and discusses the implementation of ISO guidelines in relation to acceptance or rejection of outlying values. The second aspect relates to the development of an objective statistical test for the comparison of calibration curves. Conclusion The preliminary statistical tests for outlying values and comparisons between calibration curves can be applied using basic functions found in standard spreadsheet software. These two aspects emphasise that the comparability of results arising from RT-QPCR needs further refinement and development at the data-handling phase. The implementation of standardised approaches to data analysis should further help minimise variation due to subjective judgements. The aspects described in this paper will

  18. Rapid and direct quantitative detection of viable bifidobacteria in probiotic yogurt by combination of ethidium monoazide and real-time PCR using a molecular beacon approach.

    Science.gov (United States)

    Meng, X C; Pang, R; Wang, C; Wang, L Q

    2010-11-01

    The potential of ethidium monoazide (EMA) real-time PCR method based on molecular beacon probe for rapid detection of viable bifidobacteria present in probiotic yogurt was evaluated in this work. A real-time PCR with molecular beacon assay was developed to determine genus Bifidobacterium quantitatively in order to increase the sensitivity and specificity of assay. EMA was used to treat probiotic yogurt prior to DNA extraction and real-time PCR detection to allow detection of only viable bacteria. The primer set of Bif-F/Bif-R which is genus-specific for Bifid. was designed. The specificity of the probes ensures that no signal is generated by non-target amplicons. Linear regression analysis demonstrated a good correlation (R² = 0·9948) between the EMA real-time PCR results and the plate counting, and real-time quantitative PCR results correlated adequately with enumeration of bifidobacteria by culture for commercial probiotic yogurt. This culture-independent approach is promising for the direct and rapid detection of viable bifidobacteria in commercial probiotic yogurt, and the detection can be carried out within 4 h. The detection limit for this method is about 10⁴ cell/ml. In conclusion, the direct quantitative EMA real-time PCR assay based on molecular beacon described in this research is a rapid and quantitative method.

  19. A real-time PCR-based semi-quantitative breakpoint to aid in molecular identification of urinary tract infections.

    Science.gov (United States)

    Hansen, Wendy L J; van der Donk, Christina F M; Bruggeman, Cathrien A; Stobberingh, Ellen E; Wolffs, Petra F G

    2013-01-01

    This study presents a novel approach to aid in diagnosis of urinary tract infections (UTIs). A real-time PCR assay was used to screen for culture-positive urinary specimens and to identify the causative uropathogen. Semi-quantitative breakpoints were used to screen for significant bacteriuria (presence of ≥ 10(5) CFU/ml of uropathogens) or low-level bacteriuria (containing between 10(3) and 10(4) CFU/ml of uropathogens). The 16S rDNA-based assay could identify the most prevalent uropathogens using probes for Escherichia coli, Pseudomonas species, Pseudomonas aeruginosa, Staphylococcus species, Staphylococcus aureus, Enterococcus species and Streptococcus species. 330 urinary specimens were analysed and results were compared with conventional urine culture. Using a PCR Ct value of 25 as semi-quantitative breakpoint for significant bacteriuria resulted in a sensitivity and specificity of 97% and 80%, respectively. In 78% of the samples with monomicrobial infections the assay contained probes to detect the bacteria present in the urine specimens and 99% of these uropathogens was correctly identified. Concluding, this proof-of-concept approach demonstrates that the assay can distinguish bacteriuria from no bacteriuria as well as detect the involved uropathogen within 4 hours after sampling, allowing adequate therapy decisions within the same day as well as drastically reduce consequent urine culturing.

  20. A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

    Science.gov (United States)

    Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

    2011-12-01

    Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.

  1. Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy.

    Science.gov (United States)

    Einen, Jørn; Thorseth, Ingunn H; Ovreås, Lise

    2008-05-01

    A SYBR Green real-time quantitative PCR (Q-PCR) assay for the detection and quantification of Bacteria and Archaea present in the glassy rind of seafloor basalts of different ages and water depths is presented. Two sets of domain-specific primers were designed and validated for specific detection and quantification of bacterial and archaeal 16S rRNA genes in DNA extracted from basaltic glass. Total cell numbers were also estimated by fluorescence microscopy analysis of SYBR Gold-stained samples. The results from the two different approaches were concurrent, and Q-PCR results showed that the total number of cells present in basalts was in the range from 6 x 10(5) to 4 x 10(6) cells g(-1) basaltic glass. Further, it was demonstrated that these cells were almost exclusively from the domain Bacteria. When applying the same methods on samples of different ages (22 years-0.1 Ma) and water depths (139-3390 mbsl), no significant differences in cell concentrations or in the relative abundance of Archaea and Bacteria were detected.

  2. Selection of suitable reference genes for quantitative real-time PCR in apoptosis-induced MCF-7 breast cancer cells.

    Science.gov (United States)

    Ferreira, Eloise; Cronjé, Marianne J

    2012-02-01

    Apoptosis is induced in MCF-7 breast cancer cells following treatment with salicylic acid (20 mM), either in the presence or absence of a heat shock (42°C for 30 min). In order to study the alterations of apoptotic genes with quantitative real-time PCR (qPCR), suitable genes with unchanged expression following the treatments is required for normalizing the gene expression levels. In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), Histone H2A (HIST), constitutively expressed heat shock protein 70 (HSC70) and tyrosine 3-monooxygenase/trytophan 5 monooxygenase activation protein, 14-3-3 (YWHAZ) were evaluated as appropriate reference genes. Analysis of gene expression data with one-way ANOVA, geNorm and NormFinder identified HIST and YWHAZ as the least affected during the induction of apoptosis by the different treatments, and is the most suitable gene-pair for normalization during qPCR analysis in MCF-7 breast cancer cells undergoing apoptosis following treatment with SA and/or HS.

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

    Science.gov (United States)

    Kianianmomeni, Arash; Hallmann, Armin

    2013-12-01

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

  4. Mycobacterium avium subsp. paratuberculosis survival during fermentation of soured milk products detected by culture and quantitative real time PCR methods.

    Science.gov (United States)

    Klanicova, B; Slana, I; Roubal, P; Pavlik, I; Kralik, P

    2012-07-02

    Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16 h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12 weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.

  5. Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment.

    Science.gov (United States)

    Shannon, K E; Lee, D-Y; Trevors, J T; Beaudette, L A

    2007-08-15

    Bacteria were detected at five stages of municipal wastewater treatment using TaqMan(R) real-time quantitative PCR (qPCR). Thirteen probe and primer sets were tested for diverse pathogens that may be present in wastewater, including Aeromonas hydrophila, Bacillus cereus, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, E. coli O157:H7, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella sp., and Staphylococcus aureus. The sensitivity of the assay was 100 fg of genomic DNA (=22 gene copies), based on a standard curve generated using A. hydrophila purified DNA. Samples from five stages of wastewater treatment were collected, including raw wastewater, primary effluents, mixed liquor, waste activated sludge and final effluents. In duplicate samples, E. coli, K. pneumoniae, C. perfringens and E. faecalis were detected throughout the wastewater process, and their numbers decreased by 3.52-3.98, 4.23-4.33, 3.15-3.39, and 3.24 orders of magnitude respectively, between the raw wastewater and final effluent stage. This qPCR method was effective for the detection of pathogens in wastewater and confirmed that the risk of exposure to pathogens in the wastewater discharge was well within the Environment Canada guidelines.

  6. Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera: Drosophilidae.

    Directory of Open Access Journals (Sweden)

    Yifan Zhai

    Full Text Available To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR data, normalization relative to reliable reference gene(s is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin, were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population, and abiotic (photoperiod, temperature conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper and one web-based comprehensive tool (RefFinder were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.

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

    Science.gov (United States)

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

    2013-03-01

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

  8. Use of quantitative real-time PCR for direct detection of serratia marcescens in marine and other aquatic environments.

    Science.gov (United States)

    Joyner, Jessica; Wanless, David; Sinigalliano, Christopher D; Lipp, Erin K

    2014-03-01

    Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml(-1) and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml(-1). This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  10. Quantitative detection of Borrelia burgdorferi sensu lato in erythema migrans skin lesions using internally controlled duplex real time PCR.

    Directory of Open Access Journals (Sweden)

    Maria O'Rourke

    Full Text Available B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. bavariensis are the principal species which account for Lyme borreliosis (LB globally. We have developed an internally controlled duplex quantitative real time PCR assay targeting the Borrelia 16S rRNA and the human RNAseP genes. This assay is well-suited for laboratory confirmation of suspected cases of LB and will be used to assess the efficacy of a vaccine against LB in clinical trials. The assay is highly specific, successfully detecting DNA extracted from 83 diverse B. burgdorferi sensu lato strains representing all major species causing LB, while 21 unrelated microbial species and human genomic DNA tested negative. The assay was highly reproducible and sensitive, with a lower limit of detection of 6 copies per PCR reaction. Together with culture, the assay was used to evaluate paired 3 mm skin biopsy samples taken from 121 patients presenting with solitary erythema migrans (EM lesion. PCR testing identified more positive biopsy samples than culture (77.7% PCR positive versus 55.1% culture positive and correctly identified all specimens scored as culture positive. OspA-based typing identified the majority of isolates as B. afzelii (96.8% and the bacterial load was significantly higher in culture positive biopsies than in culture negative biopsies (P<0.001. The quantitative data also enabled relationships between Borrelia burden and patient symptoms to be evaluated. The bacterial load was significantly higher among patients with systemic symptoms than without (P = 0.02 and was significantly higher for biopsies retrieved from patients with EM lesions with central clearing (P<0.001. 16S copy numbers were moderately lower in samples from patients reporting a history of LB (P = 0.10. This is the first quantitative PCR study of human skin biopsies predominantly infected with B. afzelii and the first study to demonstrate a clear relationship between clinical symptoms in B. afzelii

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

    Science.gov (United States)

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

    2017-07-01

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

  12. Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Straube Eberhard

    2010-04-01

    Full Text Available Abstract Background The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA. Methods Five kits were evaluated using clinical specimens: QIAamp™ DNA Mini Kit (QIAGEN, peqGold™ Tissue DNA Mini Kit (PeqLab, UltraClean™ Tissue and Cells DNA Isolation Kit (MoBio, DNA Isolation Kit for Cells and Tissues (Roche, and NucleoSpin™ Tissue (Macherey-Nagel. DNA yield was determined using a quantitative real-time PCR assay targeting IS711 that included an internal amplification control. Results Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p Conclusions We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.

  13. Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine▿

    Science.gov (United States)

    Hierro, Núria; Esteve-Zarzoso, Braulio; González, Ángel; Mas, Albert; Guillamón, Jose M.

    2006-01-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

  14. Accurate measurement of circulating mitochondrial DNA content from human blood samples using real-time quantitative PCR.

    Science.gov (United States)

    Ajaz, Saima; Czajka, Anna; Malik, Afshan

    2015-01-01

    We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481-492, 2013). These studies are distinct from those looking at genetic mitochondrial disease and are attempting to identify acquired changes in circulating MtDNA content as an indicator of mitochondrial function. However, the methodology being used is not always specific and reproducible. As more than 95 % of the human mitochondrial genome is duplicated in the human nuclear genome, it is important to avoid co-amplification of nuclear pseudogenes. Furthermore, template preparation protocols can also affect the results because of the size and structural differences between the mitochondrial and nuclear genomes. Here we describe how to (1) prepare DNA from blood samples; (2) pretreat the DNA to prevent dilution bias; (3) prepare dilution standards for absolute quantification using the unique primers human mitochondrial genome forward primer (hMitoF3) and human mitochondrial genome reverse primer(hMitoR3) for the mitochondrial genome, and human nuclear genome forward primer (hB2MF1) and human nuclear genome reverse primer (hB2MR1) primers for the human nuclear genome; (4) carry out qPCR for either relative or absolute quantification from test samples; (5) analyze qPCR data; and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use.

  15. Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods.

    Science.gov (United States)

    Shanks, Orin C; Kelty, Catherine A; Oshiro, Robin; Haugland, Richard A; Madi, Tania; Brooks, Lauren; Field, Katharine G; Sivaganesan, Mano

    2016-05-01

    There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria

  16. Exploring the Bacterial Diversity of Belgian Steak Tartare Using Metagenetics and Quantitative Real-Time PCR Analysis.

    Science.gov (United States)

    Delhalle, L; Korsak, N; Taminiau, B; Nezer, C; Burteau, S; Delcenserie, V; Poullet, J B; Daube, G

    2016-02-01

    Steak tartare is a popular meat dish in Belgium. It is prepared with raw minced beef and is eaten with sauce, vegetables, and spices. Because it contains raw meat, steak tartare is highly prone to bacterial spoilage. The objective of this study was to explore the diversity of bacterial flora in steak tartare in Belgium according to the source and to determine which bacteria are able to grow during shelf life. A total of 58 samples from butchers' shops, restaurants, sandwich shops, and supermarkets were collected. These samples were analyzed using 16S rDNA metagenetics, a classical microbiological technique, and quantitative real-time PCR (qPCR) targeting the Lactobacillus genus. Samples were analyzed at the beginning and at the end of their shelf life, except for those from restaurants and sandwich shops, which were analyzed only on the purchase date. Metagenetic analysis identified up to 180 bacterial species and 90 genera in some samples. But only seven bacterial species were predominant in the samples, depending on the source: Brochothrix thermosphacta, Lactobacillus algidus, Lactococcus piscium, Leuconostoc gelidum, Photobacterium kishitani, Pseudomonas spp., and Xanthomonas oryzae. With this work, an alternative method is proposed to evaluate the total flora in food samples based on the number of reads from metagenetic analysis and the results of qPCR. The degree of underestimation of aerobic plate counts at 30°C estimated with the classical microbiology method was demonstrated in comparison with the proposed culture-independent method. Compared with culture-based methods, metagenetic analysis combined with qPCR targeting Lactobacillus provides valuable information for characterizing the bacterial flora of raw meat.

  17. Reference gene selection for quantitative real-time PCR normalization in larvae of three species of Grapholitini (Lepidoptera: Tortricidae).

    Science.gov (United States)

    Ridgeway, Jaryd A; Timm, Alicia E

    2015-01-01

    Despite the agricultural importance of species in the Grapholitini (Lepidoptera: Tortricidae), and the value of gene expression analysis for improved population management, few gene expression studies based on quantitative real-time PCR (qPCR) have been conducted for this tribe. Part of the reason for this lack of information is that suitable reference genes, which are fundamental for accurate normalization of qPCR studies, have not been identified for the tribe. Thus, the expression stability of six potential reference genes (ACT, AK, COI, EF1, ENO and TUB) was assessed in three different tissues (whole body, midgut and cuticle) of Cryptophlebia peltastica (Meyrick), Cydia pomonella (L.) and Thaumatotibia leucotreta (Meyrick). Additionally, these reference genes were tested using T. leucotreta at different temperatures (15°C, 25°C and 35°C) with and without baculovirus infection. Suitable reference genes were identified for the whole body and midgut tissue of all three species, and for cuticle tissue of Cy. pomonella and T. leucotreta. When T. leucotreta was infected with the virus at all temperature conditions ACT, AK and EF1 were found to be the most suitable reference genes for experimental normalization. In general, for all tissue types, species and stress conditions, AK and EF1 were the best-performing reference genes. However, even though the three species analysed were closely related and within the same tribe, each species required varying gene combinations for suitable normalization. This study provides the first reference gene evaluation for the Tortricidae, and paves the way for future qPCR analysis in Tortricidae.

  18. Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer

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    Miller Nicola

    2007-11-01

    Full Text Available Abstract Background Real-time quantitative PCR (RQ-PCR forms the basis of many breast cancer biomarker studies and novel prognostic assays, paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC genes, used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (ESR1 was used a target gene to compare the effect of choice of EC on the estimate of gene quantity. Results The expression and validity of candidate ECs (GAPDH, TFRC, ABL, PPIA, HPRT1, RPLP0, B2M, GUSB, MRPL19, PUM1 and PSMC4 was determined in 6 benign and 21 malignant primary breast cancer tissues. Gene expression data was analysed using two different statistical models. MRPL19 and PPIA were identified as the most stable and reliable EC genes, while GUSB, RPLP0 and ABL were least stable. There was a highly significant difference in variance between ECs. ESR1 expression was appreciably higher in malignant compared to benign tissues and there was a significant effect of EC on the magnitude of the error associated with the relative quantity of ESR1. Conclusion We have validated two endogenous control genes, MRPL19 and PPIA, for RQ-PCR analysis of gene expression in primary breast tissue. Of the genes in current use in this field, the above combination offers increased accuracy and resolution in the

  19. Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

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    Peng Xia

    2009-01-01

    Full Text Available Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf mitochondrial (mtDNA and nuclear (nDNA DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

  20. Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples.

    Science.gov (United States)

    Gokduman, Kurtulus; Avsaroglu, M Dilek; Cakiris, Aris; Ustek, Duran; Gurakan, G Candan

    2016-03-01

    The aim of the current study was to develop, a new, rapid, sensitive and quantitative Salmonella detection method using a Real-Time PCR technique based on an inexpensive, easy to produce, convenient and standardized recombinant plasmid positive control. To achieve this, two recombinant plasmids were constructed as reference molecules by cloning the two most commonly used Salmonella-specific target gene regions, invA and ttrRSBC. The more rapid detection enabled by the developed method (21 h) compared to the traditional culture method (90 h) allows the quantitative evaluation of Salmonella (quantification limits of 10(1)CFU/ml and 10(0)CFU/ml for the invA target and the ttrRSBC target, respectively), as illustrated using milk samples. Three advantages illustrated by the current study demonstrate the potential of the newly developed method to be used in routine analyses in the medical, veterinary, food and water/environmental sectors: I--The method provides fast analyses including the simultaneous detection and determination of correct pathogen counts; II--The method is applicable to challenging samples, such as milk; III--The method's positive controls (recombinant plasmids) are reproducible in large quantities without the need to construct new calibration curves.

  1. Validation of reference genes for quantitative real-time PCR in Périgord black truffle (Tuber melanosporum) developmental stages.

    Science.gov (United States)

    Zarivi, Osvaldo; Cesare, Patrizia; Ragnelli, Anna Maria; Aimola, Pierpaolo; Leonardi, Marco; Bonfigli, Antonella; Colafarina, Sabrina; Poma, Anna Maria; Miranda, Michele; Pacioni, Giovanni

    2015-08-01

    The symbiotic fungus Tuber melanosporum Vittad. (Périgord black truffle) belongs to the Ascomycota and forms mutualistic symbiosis with tree and shrub roots. This truffle has a high value in a global market and is cultivated in many countries of both hemispheres. The publication of the T. melanosporum genome has given researchers unique opportunities to learn more about the biology of the fungus. Real-time quantitative PCR (qRT-PCR) is a definitive technique for quantitating differences in transcriptional gene expression levels between samples. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes must show stable expression under given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in T. melanosporum development. In this study, we present a morphological and microscopical classification of the developmental stages of T. melanosporum fruit body, and investigate the expression levels of 12 candidate reference genes (18S rRNA; 5.8S rRNA; Elongation factor 1-alpha; Elongation factor 1-beta; α-tubulin; 60S ribosomal protein L29; β-tubulin; 40S ribosomal protein S1; 40S ribosomal protein S3; Glucose-6-phosphate dehydrogenase; β-actin; Ubiquitin-conjugating enzyme). To evaluate the suitability of these genes as endogenous controls, five software-based approaches and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. We demonstrate here that the 18S rRNA gene shows the most stable expression during T. melanosporum development and that a set of three genes, 18S rRNA, Elongation factor 1-alpha and 40S ribosomal protein S3, is the most suitable to normalize qRT-PCR data from all the analyzed developmental stages; conversely, 18S rRNA, Glucose-6-phosphate dehydrogenase and Elongation factor 1-alpha are the most suitable

  2. A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR

    NARCIS (Netherlands)

    J.P.P. Meijerink (Jules); C. Mandigers; L. van de Locht; E. Tonnissen; F. Goodsaid; J. Raemaekers (John)

    2001-01-01

    textabstractQuantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been

  3. Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis

    Science.gov (United States)

    Sanchez, J. Aquiles; Pierce, Kenneth E.; Rice, John E.; Wangh, Lawrence J.

    2004-01-01

    Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)–PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80–250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CFΔ508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells. PMID:14769930

  4. 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.

  5. Quantitative real-time PCR and fluorescence in situ hybridization approaches for enumerating Brevundimonas diminuta in drinking water.

    Science.gov (United States)

    Donofrio, Robert S; Bestervelt, Lorelle L; Saha, Ratul; Bagley, Susan T

    2010-09-01

    Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B. diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100-200 bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B. diminuta were determined to be 0.89 pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B. diminuta was 1 x 10(3) colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5' 6-FAM FISH probe, traditional plating methods showed significant underestimation of B. diminuta concentration (P = 0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid (alternatives to plating when validating drinking water filtration systems.

  6. Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

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    Pek-Lan Chan

    Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection

  7. A quantitative real-time PCR assay for the identification and enumeration of Alexandrium cysts in marine sediments

    Science.gov (United States)

    Erdner, D. L.; Percy, L.; Keafer, B.; Lewis, J.; Anderson, D. M.

    2010-02-01

    Harmful algal blooms (HABs) are a global problem that affects both human and ecosystem health. One of the most serious and widespread HAB poisoning syndromes is paralytic shellfish poisoning, commonly caused by Alexandrium spp. dinoflagellates. Like many toxic dinoflagellates, Alexandrium produces resistant resting cysts as part of its life cycle. These cysts play a key role in bloom initiation and decline, as well as dispersal and colonization of new areas. Information on cyst numbers and identity is essential for understanding and predicting blooms, yet comprehensive cyst surveys are extremely time- and labor-intensive. Here we describe the development and validation of a quantitative real-time PCR (qPCR) technique for the enumeration of cysts of A. tamarense of the toxic North American/Group I ribotype. The method uses a cloned fragment of the large subunit ribosomal RNA gene as a standard for cyst quantification, with an experimentally determined conversion factor of 28,402±6152 LSU ribosomal gene copies per cyst. Tests of DNA extraction and PCR efficiency show that mechanical breakage is required for adequate cyst lysis, and that it was necessary to dilute our DNA extracts 50-fold in order to abolish PCR inhibition from compounds co-extracted from the sediment. The resulting assay shows a linear response over 6 orders of magnitude and can reliably quantify ≥10 cysts/cm 3 sediment. For method validation, 129 natural sediment samples were split and analyzed in parallel, using both the qPCR and primulin-staining techniques. Overall, there is a significant correlation ( p<0.001) between the cyst abundances determined by the two methods, although the qPCR counts tend to be lower than the primulin values. This underestimation is less pronounced in those samples collected from the top 1 cm of sediment, and more pronounced in those derived from the next 1-3 cm of the core. These differences may be due to the condition of the cysts in the different layers, as the

  8. Quantitative analysis of herpes virus sequences from normal tissue and fibropapillomas of marine turtles with real-time PCR

    Science.gov (United States)

    Quackenbush, S.L.; Casey, R.N.; Murcek, R.J.; Paul, T.A.; Work, T.M.; Limpus, C.J.; Chaves, A.; duToit, L.; Perez, J.V.; Aguirre, A.A.; Spraker, T.R.; Horrocks, J.A.; Vermeer, L.A.; Balazs, G.S.; Casey, J.W.

    2001-01-01

    Quantitative real-time PCR has been used to measure fibropapilloma-associated turtle herpesvirus (FPTHV) pol DNA loads in fibropapillomas, fibromas, and uninvolved tissues of green, loggerhead, and olive ridley turtles from Hawaii, Florida, Costa Rica, Australia, Mexico, and the West Indies. The viral DNA loads from tumors obtained from terminal animals were relatively homogenous (range 2a??20 copies/cell), whereas DNA copy numbers from biopsied tumors and skin of otherwise healthy turtles displayed a wide variation (range 0.001a??170 copies/cell) and may reflect the stage of tumor development. FPTHV DNA loads in tumors were 2.5a??4.5 logs higher than in uninvolved skin from the same animal regardless of geographic location, further implying a role for FPTHV in the etiology of fibropapillomatosis. Although FPTHV pol sequences amplified from tumors are highly related to each other, single signature amino acid substitutions distinguish the Australia/Hawaii, Mexico/Costa Rica, and Florida/Caribbean groups.

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

    Directory of Open Access Journals (Sweden)

    Christoph F

    2006-03-01

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

  10. Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Kawaguchi Makoto

    2010-01-01

    Full Text Available Abstract Background Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD, squamous cell carcinoma (SQ, large cell carcinoma (LC, and small cell carcinoma (SC. Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR. Results We selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA and a normal control lung cell line (MRC-9. From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L. Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2. The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression. Conclusions These results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results

  11. Validation of RNAi by real time PCR

    DEFF Research Database (Denmark)

    Josefsen, Knud; Lee, Ying Chiu

    2011-01-01

    Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to veri...

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

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    Hui Ling

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

  13. Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples.

    Science.gov (United States)

    Irenge, Léonid M; Durant, Jean-François; Tomaso, Herbert; Pilo, Paola; Olsen, Jaran S; Ramisse, Vincent; Mahillon, Jacques; Gala, Jean-Luc

    2010-11-01

    A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

  14. Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar(®) HHV-6 PCR Kit for quantitative detection of human herpesvirus 6.

    Science.gov (United States)

    Yip, Cyril C Y; Sridhar, Siddharth; Cheng, Andrew K W; Fung, Ami M Y; Cheng, Vincent C C; Chan, Kwok-Hung; Yuen, Kwok-Yung

    2017-08-01

    HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar(®) HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar(®) HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log10 copies/ml and a coefficient of determination (R(2)) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log10 and 2 log10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar(®) HHV-6 PCR Kit (R(2)=0.926; PPCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR

    Science.gov (United States)

    Droplet digital Polymerase chain reaction (ddPCR) is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. It is a promising DNA quantification technology for medical diagnostics but there are only a few reports of its use for plant pat...

  16. Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

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    Li Qingdi

    2012-06-01

    Full Text Available Abstract Background The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. Results The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25 remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4 were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13 as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such

  17. Selection and validation of appropriate reference genes for quantitative real-time PCR analysis of gene expression in Lycoris aurea

    Directory of Open Access Journals (Sweden)

    Rui eMa

    2016-04-01

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

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  19. Identification of stable reference genes for gene expression studies using quantitative real time PCR in buffalo oocytes and embryos.

    Science.gov (United States)

    Kumar, Parveen; Yadav, Poonam; Verma, Arpana; Singh, Dheer; De, Sachinandan; Datta, Tirtha Kumar

    2012-12-01

    The present study was aimed to validate expression stability of 6 housekeeping genes (viz. YWHAZ, SDHA, GAPDH, RPS15, RPS18 and RN18S1) in the oocytes and embryos of different stages in buffalo. A modified Trizol protocol was optimized for RNA isolation from as few as five oocytes. The expression level of selected genes was studied by an optimized real time PCR using DCT method and their stability of expression was evaluated by Microsoft Excel based visual application, geNORM. The analysis revealed that the RPS15 and GAPDH were the most stable genes across different samples. Also, the geometric mean of three genes (i.e. RPS15,RPS18 and GAPDH) were found suitable for normalization of real time PCR data from buffalo oocytes⁄embryos. The information would help in more accurate interpretation of gene expression data from oocytes⁄embryos towards understanding the molecular events in these cells during development.

  20. Development of Quantitative Competitive PCR and Absolute Based Real-Time PCR Assays for Quantification of The Butyrate Producing Bacterium: Butyrivibrio fibrisolvens

    Directory of Open Access Journals (Sweden)

    Mojtaba Tahmoorespur

    2016-04-01

    Full Text Available Introduction Butyrivibrio fibrisolvens strains are presently recognized as the major butyrate-producing bacteria found in the rumen and digestive track of many animals and also in the human gut. In this study we reported the development of two DNA based techniques, quantitative competitive (QC PCR and absolute based Real-Time PCR, for enumerating Butyrivibrio fibrisolvens strains. Despite the recent introduction of real-time PCR method for the rapid quantification of the target DNA sequences, use of quantitative competitive PCR (QC-PCR technique continues to play an important role in nucleic acid quantification since it is more cost effective. The procedure relies on the co-amplification of the sequence of interest with a serially diluted synthetic DNA fragment of the known concentration (competitor, using the single set primers. A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR. It monitors the amplification of a targeted DNA molecule during the PCR. Materials and Methods At first reported species-specific primers targeting the 16S rDNA region of the bacterium Butyrivibrio fibrisolvens were used for amplifying a 213 bp fragment. A DNA competitor differing by 50 bp in length from the 213 bp fragment was constructed and cloned into pTZ57R/T vector. The competitor was quantified by NanoDrop spectrophotometer and serially diluted and co-amplified by PCR with total extracted DNA from rumen fluid samples. PCR products were quantified by photographing agarose gels and analyzed with Image J software and the amount of amplified target DNA was log plotted against the amount of amplified competitor. Coefficient of determination (R2 was used as a criterion of methodology precision. For developing the Real-time PCR technique, the 213 bp fragment was amplified and cloned into pTZ57R/T was used to draw a standard curve. Results and Discussion The specific primers of Butyrivibrio

  1. Quantitative detection of Cucumber vein yellowing virus in susceptible and partially resistant plants using real-time PCR.

    Science.gov (United States)

    Picó, Belén; Sifres, Alicia; Nuez, Fernando

    2005-09-01

    A method for the detection of Cucumber vein yellowing virus (CVYV) that combines reverse transcription with real-time PCR (SYBR((R)) Green chemistry) was developed using specific primers designed from a nucleotide sequence of the RNA polymerase gene (NIb) conserved among all the available CVYV strains. This method provided a linear assay over five to six orders of magnitude and reproducibly detected titres as low as 10(3) molecules of the target CVYV cDNA. Real-time PCR gave reproducible results for the quantification of CVYV in young leaves of susceptible and resistant cucumber landraces after mechanical inoculation. Significant differences in the starting amount of target cDNA were found between the analyzed genotypes, indicating differences in viral accumulation that correlated to their different levels of resistance. Real-time PCR results validated our previous findings using slot-blot hybridization, the dominance of the strong resistance to CVYV displayed by C.sat 10, and provided improved reliability and sensitivity of detection. This method has great potential in resistance breeding for germplasm screening, characterization of resistance mechanisms and genetic studies.

  2. Effect of carbon monoxide on gene expression in cerebrocortical astrocytes: Validation of reference genes for quantitative real-time PCR.

    Science.gov (United States)

    Oliveira, Sara R; Vieira, Helena L A; Duarte, Carlos B

    2015-09-15

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as cytoprotection or inflammation. The accurate assessment of changes in gene expression depends on the selection of adequate internal reference gene(s). Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to this gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of CO on gene expression in cultured cerebrocortical astrocytes. The cells were exposed to CO by treatment with CORM-A1 (CO releasing molecule A1) and four different algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to evaluate the stability of eight putative reference genes. Our results indicate that Gapdh (glyceraldehyde-3-phosphate dehydrogenase) together with Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha (Succinate Dehydrogenase Complex, Subunit A), Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-A1 for up to 60 min. For validation, we analyzed the effect of CO on the expression of Bdnf and bcl-2. Different results were obtained, depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. These findings highlight the need for a proper and accurate selection of the reference genes used in the quantification of qRT-PCR results

  3. Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions.

    Science.gov (United States)

    Zhu, Jianfeng; Zhang, Lifeng; Li, Wanfeng; Han, Suying; Yang, Wenhua; Qi, Liwang

    2013-01-01

    Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1α would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1α were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana.

  4. Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions.

    Directory of Open Access Journals (Sweden)

    Jianfeng Zhu

    Full Text Available Quantitative real-time reverse transcription polymerase chain reaction (qPCR, a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat stresses, in two distinct plant organs (roots and leaves. The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1α would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1α were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2 were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana.

  5. Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.

    Directory of Open Access Journals (Sweden)

    Qiusheng Kong

    Full Text Available Melon (Cucumis melo. L is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR, which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.

  6. Fibroblast growth factor receptor 1 amplification in non-small cell lung cancer by quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Shirish M Gadgeel

    Full Text Available INTRODUCTION: Amplification of the fibroblast growth factor receptor 1 (FGFR1 gene has been described in tumors of non-small-cell lung cancer (NSCLC patients. Prior reports showed conflicting rates of amplification frequency and clinical relevance. MATERIALS AND METHODS: We developed a reliable real-time quantitative PCR assay to assess the frequency of FGFR1 amplification and assessed the optimal cutoff level of amplification for clinical application. RESULTS: In a training cohort of 203 NSCLCs, we established that a 3.5-fold amplification optimally divided patients into groups with different survival rates with a clear threshold level. Those with FGFR1 amplification levels above 3.5-fold had an inferior survival. These data were confirmed in a validation cohort of 142 NSCLC. After adjusting for age, sex, performance status, stage, and histology, patients with FGFR1 amplification levels above 3.5 fold had a hazard ratio of 2.91 (95% CI- 1.14, 7.41; pvalue-0.025 for death in the validation cohort. The rates of FGFR1 amplification using the cutoff level of 3.5 were 5.1% in squamous cell and 4.1% in adenocarcinomas. There was a non-significant trend towards higher amplifications rates in heavy smokers (> 15 pack-years of cigarette consumption as compared to light smokers. DISCUSSION: Our data suggest that a 3.5-fold amplification of FGFR1 is of clinical importance in NSCLC. Our cutpoint analysis showed a clear threshold effect for the impact of FGFR1 amplification on patients' survival, which can be used as an initial guide for patient selection in trials assessing efficacy of novel FGFR inhibitors.

  7. Systematic evaluation of three microRNA profiling platforms: microarray, beads array, and quantitative real-time PCR array.

    Science.gov (United States)

    Wang, Bin; Howel, Paul; Bruheim, Skjalg; Ju, Jingfang; Owen, Laurie B; Fodstad, Oystein; Xi, Yaguang

    2011-02-11

    A number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA) microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA). The microRNA profiles of 40 human osteosarcoma xenograft samples were generated by LNA array, beads array, and TLDA. Results show that each of the three platforms perform similarly regarding intra-platform reproducibility or reproducibility of data within one platform while LNA array and TLDA had the best inter-platform reproducibility or reproducibility of data across platforms. The endogenous controls/probes contained in each platform have been observed for their stability under different treatments/environments; those included in TLDA have the best performance with minimal coefficients of variation. Importantly, we identify that the proper selection of normalization methods is critical for improving the inter-platform reproducibility, which is evidenced by the application of two non-linear normalization methods (loess and quantile) that substantially elevated the sensitivity and specificity of the statistical data assessment. Each platform is relatively stable in terms of its own microRNA profiling intra-reproducibility; however, the inter-platform reproducibility among different platforms is low. More microRNA specific normalization methods are in demand for cross-platform microRNA microarray data integration and comparison, which will improve the reproducibility and consistency between platforms.

  8. Detecting mRNA Predictors of Acetaminophen-Induced Hepatotoxicity in Mouse Blood Using Quantitative Real-Time PCR.

    Science.gov (United States)

    Kanno, Syu-ichi; Tomizawa, Ayako; Yomogida, Shin

    2016-01-01

    Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. Drug-induced liver injury from agents such as APAP is known to vary between individuals within a species. To avoid liver injury and ensure the proper use of pharmaceutical products, it is important to be able to predict such risks using genetic information. This study evaluated the use of quantitative real-time polymerase chain reaction (RT-qPCR) to identify mRNAs (carried in the blood of male ddY mice) capable of predicting susceptibility to APAP-induced hepatotoxicity. Screening was performed on samples obtained at 18 h after treatment from mice that had been orally treated with 500 mg/kg APAP. APAP-induced hepatotoxicity was seen in 60% of the mice, and the mortality rate was 12%. Blood APAP concentration did not differ significantly between mice with and without APAP-induced hepatotoxicity. We compared blood mRNA expression levels between mice with (positive, serious or lethal injury) and without hepatotoxicity in the APAP-treated group. The transcript levels of interleukin-encoding loci Il1β, Il10, and tumor necrosis factor (Tnf) were increased in the lethal injury group. Transcripts of the loci encoding transthyretin (Ttr) and metallothionein 1 (Mt1) showed increases in the liver injury group, while those of the glutathione peroxidase 3-encoding locus (Gpx3) were decreased. APAP hepatotoxicity was potentiated in fasted animals, although fasting did not appear to affect the level of expression of these genes. These results indicate that mRNA expression of Il1β, Il10, Tnf, Ttr, Mt1, and Gpx3 in mouse blood may provide useful surrogate markers of APAP-induced hepatotoxicity.

  9. Limitations of the use of group-specific primers in real-time PCR as appear from quantitative analyses of closely related ammonia-oxidising species

    NARCIS (Netherlands)

    Sekido, T.; Bodelier, P.L.E.; Shoji, T.; Suwa, Y.; Laanbroek, R.

    2008-01-01

    To study the ecology of ammonia-oxidising bacteria (AOB), quantitative techniques are essential. Real-time PCR assays based on the 16S rRNA or on the structural amoA gene are routinely used. The CTO primer set rooted on the 16S rRNA gene has a number of mismatches with some of the cultures of AOB. T

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

    Science.gov (United States)

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

  11. Limitations of the use of group-specific primers in real-time PCR as appear from quantitative analyses of closely related ammonia-oxidising species

    NARCIS (Netherlands)

    Sekido, T.; Bodelier, P.L.E.; Shoji, T.; Suwa, Y.; Laanbroek, R.

    2008-01-01

    To study the ecology of ammonia-oxidising bacteria (AOB), quantitative techniques are essential. Real-time PCR assays based on the 16S rRNA or on the structural amoA gene are routinely used. The CTO primer set rooted on the 16S rRNA gene has a number of mismatches with some of the cultures of AOB. T

  12. MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments

    NARCIS (Netherlands)

    Bustin, S.A.; Beaulieu, J.F.; Huggett, J.; Jaggi, R.; Kibenge, F.S.; Olsvik, P.A.; Penning, L.C.; Toegel, S.

    2010-01-01

    MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments Stephen A Bustin1 , Jean-François Beaulieu2 , Jim Huggett3 , Rolf Jaggi4 , Frederick SB Kibenge5 , Pål A Olsvik6 , Louis C Penning7 and Stefan Toegel8 1 Centre for Diges

  13. Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR.

    Science.gov (United States)

    Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes.

  14. Real Time PCR: Principles and Application

    Directory of Open Access Journals (Sweden)

    Safie Amini

    2005-09-01

    levels are compared between samples, the expression level of the target is referred to as being, for example, 100-fold higher in stimulated cells than in unstimulated cells. The quantification procedure differs depending on whether the target and the endogenous reference genes are amplified with comparable or different efficiencies(20, 21.Viral QuantificationThe possibility to detect viral DNA or RNA in a quantitative manner has already contributed significantly to the management and diagnosis of viral infections, as well as to the understanding of virus-host interactions. New developments in amplification techniques based on real-time detection, as well as automation of the whole process, will soon be introduced in a diagnostic laboratory setting, thereby enabling a rapid turnaround time to generate both quantitative and qualitative results. The development of molecular techniques that access viral load and the development of genotypic resistance have revolutionized the treatment of HIV disease. Commercially available viral load assays use a number of different approaches from reverse transcriptase PCR to branched DNA signalamplification. The drawbacks of the assay are that there is no international standard that allows comparison of viral load between assays and that the diversity of different clades of HIV results in under or the nondetection of some patients' samples. New real-time PCR assays are under development, including LightCycler- and TaqMan-based tests. The development of sequence-based genotyping assays for the detection of mutations associated with the development of the resistance to the 17 licensed drugs targeted against the pol gene of HIV have added to the improvements in patient management. However, next-generation assays must extend detection to include the gp41 fusion region and the integrase region of the genome as compounds directed against these targets move from clinical trails into licensed drugs. Also, genotypic assays must improve detection of

  15. Quantitative Detection of Clostridium perfringens in Broiler Chickens by Real-Time PCR Targeting the Alpha-Toxin Gene

    DEFF Research Database (Denmark)

    Abildgaard, Lone; Engberg, Ricarda M.; Schramm, Andreas

    2006-01-01

    was developed by sequencing the α-toxin gene from ~60 strains of C. perfringens, isolated from diseased as well as healthy broilers. For its application to the chicken gastrointestinal tract (i.e., ileum), DNA extraction efficiency and potential inhibition of the real-time PCR process by ileum content...... was evaluated by (i) analysing ileum samples spiked with C. perfringens cells, and (ii) comparing DNA extractions/real-time PCR of pure cultures with and without ileum content added. No inhibition was observed, and the quantitative real-time PCR approach correlated well with counts in pure cultures and spiked...... ileum content. The protocol had a detection limit of ~1000 cells spiked into 200 mg ileal material. This matched the sensitivity obtained using published primers targeting C. perfringens 16S rDNA. However, targeting the functional α-toxin gene offers the possibility to assess not only abundance of C...

  16. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

    Directory of Open Access Journals (Sweden)

    Akiko Edagawa

    2015-10-01

    Full Text Available We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR, and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%. Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%. In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8% compared with real-time qPCR alone (46/68, 67.6%. Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1% compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%. Legionella was not detected in the remaining six samples (6/68, 8.8%, irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  17. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.

    Science.gov (United States)

    Edagawa, Akiko; Kimura, Akio; Kawabuchi-Kurata, Takako; Adachi, Shinichi; Furuhata, Katsunori; Miyamoto, Hiroshi

    2015-10-19

    We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  18. Validation of a quantitative real-time PCR assay for HTLV-1 proviral load in peripheral blood mononuclear cells.

    Science.gov (United States)

    Rosadas, Carolina; Cabral-Castro, Mauro Jorge; Vicente, Ana Carolina Paulo; Peralta, José Mauro; Puccioni-Sohler, Marzia

    2013-11-01

    The objective of this study was to validate a TaqMan real-time PCR assay for HTLV-1 proviral load detection in peripheral blood mononuclear cells. TARL-2 cells were used to generate a standard curve. Peripheral blood mononuclear cell gDNA from 27 seropositive and 23 seronegative samples was analyzed. The sensitivity, specificity, accuracy, precision, dynamic range of the standard curve and qPCR efficiency were evaluated. All of the positive samples amplified the target gene. All of the negative samples amplified only the control gene (β-actin). The assay presented 100% specificity and sensibility. The intra- and inter-assay variability was 2.4% and 2.2%, respectively. The qPCR efficiency, slope and correlation coefficients (r2) were all acceptable. The limit of detection was 1 copy/rxn. This assay can reliably quantify HTLV-1 proviral load.

  19. 实时定量PCR技术的介绍%Introduction of real-time quantitative PCR technology

    Institute of Scientific and Technical Information of China (English)

    张立国; 张琚

    2003-01-01

    @@ 实时定量PCR(real-time PCR)技术是近几年发展起来的新技术,既保持了PCR技术灵敏、快速的特点,又克服了以往PCR技术中存在的假阳性污染和不能进行准确定量的缺点.另外,还有重复性好、省力、低费用等优点.实时定量PCR技术是从传统PCR技术发展而来,其基本原理是相同的,主要不同之处是其定量的体系.下面简单介绍一下该技术定量的原理.

  20. Real-time quantitative reverse transcription-PCR analysis of expression stability of Actinobacillus pleuropneumoniae housekeeping genes during in vitro growth under iron-depleted conditions

    DEFF Research Database (Denmark)

    Nielsen, K. K.; Boye, Mette

    2005-01-01

    The aims of the present investigation were to develop and test a sensitive and reproducible method for the study of gene expression in the porcine lung pathogen Actinobacillus pleuropneumoniae by real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal...... up-regulation under iron-restricted conditions compared to bacteria grown in medium with sufficient iron. The observed expression patterns of the genes of interest were consistent with previous observations. This study therefore lends further support to the use of real-time quantitative RT...

  1. Novel quantitative real-time PCR approach to determine safflower (Carthamus tinctorius) adulteration in saffron (Crocus sativus).

    Science.gov (United States)

    Villa, Caterina; Costa, Joana; Oliveira, M Beatriz P P; Mafra, Isabel

    2017-08-15

    This work intended to develop DNA-based methods to detect and quantify safflower as an adulterant in saffron. Species-specific PCR and real-time PCR with EvaGreen dye targeting the ITS region of Carthamus tinctorius L. (safflower) were successfully proposed. The assays allowed absolute and relative sensitivities of 2pg of safflower DNA (∼1.4 DNA copies) and 0.1% of safflower in saffron (Crocus sativus L.), respectively. A normalised real-time PCR approach was also proposed in the range of 0.1-20% (w/w) of safflower in saffron, which was successfully validated and applied to commercial saffron samples (stigmas, powders and seasonings). From 19 samples, three were positive to safflower, though at levels below the limit of detection, suggesting cross-contamination rather than adulteration. In this work, specific, sensitive and accurate tools were proposed to authenticate saffron. To the best of our knowledge, this is the first successful attempt to quantify safflower by a DNA-based approach.

  2. Development of real-time immuno-PCR for the quantitative detection of mycobacterial PstS1 in tuberculosis patients.

    Science.gov (United States)

    Sharma, Suman; Raj, Ankush; Singh, Netrapal; Dahiya, Bhawna; Sheoran, Abhishek; Gupta, Krishna B; Mehta, Promod K

    2017-01-01

    A novel indirect real-time immuno-polymerase chain reaction (RT-I-PCR) assay, an evolution of I-PCR, was developed for the quantitative detection of Mycobacterium tuberculosis PstS1 (Rv0934) with a wide dynamic range of 10ng/mL to 1pg/mL in body fluids of tuberculosis (TB) patients, which may monitor the dynamics of disease. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction based on msp5 gene

    Directory of Open Access Journals (Sweden)

    Gisele M. Bacanelli

    2014-01-01

    Full Text Available The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

  4. Real-time PCR: Advanced technologies and applications

    Science.gov (United States)

    This book brings together contributions from 20 experts in the field of PCR, providing a broad perspective of the applications of quantitative real-time PCR (qPCR). The editors state in the preface that the aim is to provide detailed insight into underlying principles and methods of qPCR to provide ...

  5. Analysis of Gene and Protein Expression in Atherosclerotic Mouse Aorta by Western Blot and Quantitative Real-Time PCR.

    Science.gov (United States)

    Rivera-Torres, José

    2015-01-01

    Atherosclerosis involves changes in gene and protein expression patterns in affected arteries. Quantification of these alterations is essential for understanding the molecular mechanisms underlying this pathology. Western blot and real-time PCR-used to quantify protein and messenger RNA levels, respectively-are invaluable molecular biology tools, particularly when material is limited. The availability of many genetically modified mouse models of atherosclerosis makes the mouse aorta an ideal tissue in which to carry out these expression pattern analyses. In this chapter, protocols are presented for mRNA and protein extraction from mouse aorta and for the accurate quantification of mRNA expression by RT-PCR and of proteins by western blot.

  6. Quantitative detection of pork in commercial meat products by TaqMan® real-time PCR assay targeting the mitochondrial D-loop region.

    Science.gov (United States)

    Kim, Miju; Yoo, Insuk; Lee, Shin-Young; Hong, Yeun; Kim, Hae-Yeong

    2016-11-01

    The TaqMan® real-time PCR assay using the mitochondrial D-loop region was developed for the quantitative detection of pork in processed meat products. The newly designed primers and probe specifically amplified pork without any cross-reactivity with non-target animal species. The limit of detection of the real-time PCR assay was 0.1pg of heat-treated pork meat and 0.1% (w/w) pork meat in beef and chicken meat mixtures. The quantitative real-time PCR assay was applied to analyze the pork meat content in 22 commercial processed meat products including jerkies, press hams, sausages, hamburger patties and steaks, grilled short rib patties, and nuggets. The developed real-time PCR method was able to detect pork meat in various types of processed meat products that declared the use of pork meat on their label. All processed meat products that declared no use of pork meat showed a negative result in the assay. The method developed in this study showed sensitivity and specificity in the quantification of pork meat in commercial processed meat products.

  7. Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues

    Directory of Open Access Journals (Sweden)

    Riond Barbara

    2009-12-01

    Full Text Available Abstract Background Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan® real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats. Results RNA extraction from tissues was optimised for minimal genomic DNA (gDNA contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL, β-actin (ACTB, β-2-microglobulin (B2M, β-glucuronidase (GUSB, hydroxymethyl-bilane synthase (HMBS, hypoxanthine phosphoribosyltransferase (HPRT, ribosomal protein S7 (RPS7, and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ. The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ. The assays were tested together with previously developed TaqMan® assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA and the least abundant genes (ABL, GUSB, and HMBS. The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the

  8. Quantitative multiplex real-time PCR assay for shrimp allergen: comparison of commercial master mixes and PCR platforms in rapid cycling.

    Science.gov (United States)

    Eischeid, Anne C; Kasko, Sasha M

    2015-01-01

    Real-time PCR has been used widely in numerous fields. In food safety, it has been applied to detection of microbes and other contaminants, including food allergens. Interest in rapid (fast) cycling real-time PCR has grown because it yields results in less time than does conventional cycling. However, fast cycling can adversely affect assay performance. Here we report on tests of commercial master mixes specifically designed for fast real-time PCR using a shrimp allergen assay we previously developed and validated. The objective of this work was to determine whether specialized commercial master mixes lead to improved assay performance in rapid cycling. Real-time PCR assays were carried out using four different master mixes and two different rapid cycling protocols. Results indicated that specialized master mixes did yield quality results. In many cases, linear ranges spanned up to 7 orders of magnitude, R(2) values were at least 0.95, and reaction efficiencies were within or near the optimal range of 90 to 110%. In the faster of the two rapid cycling protocols tested, assay performance and PCR amplification were markedly better for the shorter PCR product. In conclusion, specialized commercial master mixes were effective as part of rapid cycling protocols, but conventional cycling as used in our previous work is more reliable for the shrimp assay tested.

  9. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    . In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical...

  10. Validation of a carnation-specific gene, ANS, used as an endogenous reference gene in qualitative and real-time quantitative PCR for carnations.

    Science.gov (United States)

    Zhu, Hong; Jiang, Lingxi; Tao, Shiru; Lin, Heyan; Wang, Jinbin; Tan, Furong; Zhao, Kai; Wu, Xiao; Li, Peng; Pan, Aihu; Jia, Junwei; Tang, Xueming

    2011-01-01

    The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modified (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplification products were obtained with all of them. No amplification products were observed with samples from 14 other plant species, which demonstrated that the system was specific to carnation. The results of Southern blot analysis confirmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR efficiency and linearity. Thus, the ANS gene had species specificity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.

  11. Detection of Apoptotic Inhibitor Gene Survivin in Peripheral Blood of Patients with Esophageal Cancer by Real-time Fluorescence Quantitative PCR and its Clinical Signiifcance

    Institute of Scientific and Technical Information of China (English)

    CHEN Sheng

    2014-01-01

    Objective:To explore the clinical signiifcance of apoptotic inhibitor gene Survivin in peripheral blood of patients with esophageal cancer. Methods:Real-time lfuorescence quantitative PCR was used to detect the expression of Survivin mRNA in peripheral blood of 93 patients with benign and malignant esophageal lesions. The relationship of Survivin mRNA expression and clinicopathologic feature was observed. Results:The expression of Survivin mRNA in peripheral blood which was associated with differentiated degree and clinical staging was progressively increased from benign lesion to carcinoma in situ and invasive carcinoma. Conclusion:The expression of Survivin mRNA in peripheral blood is significantly related to the genesis and progression of esophageal carcinoma. Real-time fluorescence quantitative PCR used to detect the expression of Survivin m-RNA in peripheral blood may be more convenient for diagnosing and guiding the treatment of esophageal carcinoma.

  12. PROSPECTIVE STUDY OF MULTIPLE GENETIC TUMOR MARKER ASSAY BY QUANTITATIVE REAL-TIME PCR TO PREDICT RECURRENCE IN COLORECTAL CANCER PATIENTS

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Objective To describe correlation between multiple genetic tumor markers,carcinoembryonic antigen (CEA),cytokeratin 20 (CK20),and Survivin,and clinicopathological features of colorectal cancer (CRC) and to assess prognostic diagnosis value in cancer recurrence and metastasis.Methods A total of 92 patients with CRC,68 patients with precancerous lesions,and 29 control volunteers were collected for the detection of CEA,CK20,and Survivin expressions by using quantitative Real-Time PCR technology.Associations am...

  13. Rapid Diagnosis of Mycobacterial Infections and Quantitation of Mycobacterium tuberculosis Load by Two Real-Time Calibrated PCR Assays

    Science.gov (United States)

    Broccolo, Francesco; Scarpellini, Paolo; Locatelli, Giuseppe; Zingale, Anna; Brambilla, Anna M.; Cichero, Paola; Sechi, Leonardo A.; Lazzarin, Adriano; Lusso, Paolo; Malnati, Mauro S.

    2003-01-01

    Sensitive and specific techniques to detect and identify Mycobacterium tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (TB). We developed two real-time PCR assays, based on the IS6110 multicopy element and on the senX3-regX3 intergenic region, which provide a rapid method for the diagnosis of mycobacterial infections. The sensitivity and specificity of both assays were established by using purified DNA from 71 clinical isolates and 121 clinical samples collected from 83 patients, 20 of whom were affected by TB. Both assays are accurate, sensitive, and specific, showing a complementary pattern of Mycobacterium recognition: broader for the IS6110-based assay and restricted to the M. tuberculosis complex for the senX3-regX3-based assay. Moreover, the addition of a synthetic DNA calibrator prior to DNA extraction allowed us to measure the efficiency of DNA recovery and to control for the presence of PCR inhibitors. The mycobacterial burden of the clinical samples, as assessed by direct microscopy, correlates with the M. tuberculosis DNA load measured by the senX3-regX3-based assay. In addition, reduced levels of M. tuberculosis DNA load are present in those patients subjected to successful therapy, suggesting a potential use of this assay for monitoring treatment efficacy. Therefore, these assays represent a fully controlled high-throughput system for the evaluation of mycobacterial burden in clinical specimens. PMID:14532183

  14. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    Science.gov (United States)

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis.

  15. LEMming: A Linear Error Model to Normalize Parallel Quantitative Real-Time PCR (qPCR) Data as an Alternative to Reference Gene Based Methods.

    Science.gov (United States)

    Feuer, Ronny; Vlaic, Sebastian; Arlt, Janine; Sawodny, Oliver; Dahmen, Uta; Zanger, Ulrich M; Thomas, Maria

    2015-01-01

    Gene expression analysis is an essential part of biological and medical investigations. Quantitative real-time PCR (qPCR) is characterized with excellent sensitivity, dynamic range, reproducibility and is still regarded to be the gold standard for quantifying transcripts abundance. Parallelization of qPCR such as by microfluidic Taqman Fluidigm Biomark Platform enables evaluation of multiple transcripts in samples treated under various conditions. Despite advanced technologies, correct evaluation of the measurements remains challenging. Most widely used methods for evaluating or calculating gene expression data include geNorm and ΔΔCt, respectively. They rely on one or several stable reference genes (RGs) for normalization, thus potentially causing biased results. We therefore applied multivariable regression with a tailored error model to overcome the necessity of stable RGs. We developed a RG independent data normalization approach based on a tailored linear error model for parallel qPCR data, called LEMming. It uses the assumption that the mean Ct values within samples of similarly treated groups are equal. Performance of LEMming was evaluated in three data sets with different stability patterns of RGs and compared to the results of geNorm normalization. Data set 1 showed that both methods gave similar results if stable RGs are available. Data set 2 included RGs which are stable according to geNorm criteria, but became differentially expressed in normalized data evaluated by a t-test. geNorm-normalized data showed an effect of a shifted mean per gene per condition whereas LEMming-normalized data did not. Comparing the decrease of standard deviation from raw data to geNorm and to LEMming, the latter was superior. In data set 3 according to geNorm calculated average expression stability and pairwise variation, stable RGs were available, but t-tests of raw data contradicted this. Normalization with RGs resulted in distorted data contradicting literature, while

  16. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD....... In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical...

  17. Selection of Reference Genes for Quantitative Real-time PCR during Flower Development in Tree Peony (Paeonia suffruticosa Andr.

    Directory of Open Access Journals (Sweden)

    Jian eLi

    2016-04-01

    Full Text Available Tree peony (Paeonia suffruticosa is a perennial plant indigenous to China known for its elegant and vibrantly colorful flowers. A few genes involved in petal pigmentation have been cloned in tree peony. However, to date, there have been few studies on the comparison and selection of stable reference genes for gene expression analysis by quantitative reverse-transcription PCR (qRT-PCR in this species. In this study, 10 candidate reference genes were evaluated for the normalization of qRT-PCR in three tree peony cultivars. GAPDH and UBC were identified as the top two most stable reference genes in ‘Feng Dan’ and ‘Xi Shi’, and EF-1α/UBC was recommended to be the best combination for ‘Que Hao’. The expression stability of various reference genes differed across cultivars, suggesting that selection and validation of reliable reference genes for quantitative gene expression analysis was necessary not only for different species but also for different cultivars. The results provided a list of reference genes for further study on gene expression in P. suffruticosa. However, in any case, a preliminary check on the accuracy of the best performing reference genes is requested for each qRT-PCR experiment.

  18. Selection of Reference Genes for Quantitative Real-Time PCR during Flower Development in Tree Peony (Paeonia suffruticosa Andr.)

    Science.gov (United States)

    Li, Jian; Han, Jigang; Hu, Yonghong; Yang, Ji

    2016-01-01

    Tree peony (Paeonia suffruticosa) is a perennial plant indigenous to China known for its elegant and vibrantly colorful flowers. A few genes involved in petal pigmentation have been cloned in tree peony. However, to date, there have been few studies on the comparison and selection of stable reference genes for gene expression analysis by quantitative reverse-transcription PCR (qRT-PCR) in this species. In this study, 10 candidate reference genes were evaluated for the normalization of qRT-PCR in three tree peony cultivars. GAPDH and UBC were identified as the top two most stable reference genes in ‘Feng Dan’ and ‘Xi Shi,’ and EF-1α/UBC was recommended to be the best combination for ‘Que Hao.’ The expression stability of various reference genes differed across cultivars, suggesting that selection and validation of reliable reference genes for quantitative gene expression analysis was necessary not only for different species but also for different cultivars. The results provided a list of reference genes for further study on gene expression in P. suffruticosa. However, in any case, a preliminary check on the accuracy of the best performing reference genes is requested for each qRT-PCR experiment. PMID:27148337

  19. Real time PCR. Application in dengue studies

    Directory of Open Access Journals (Sweden)

    Jeanette Prada-Arismendy

    2011-06-01

    Full Text Available PCR (polymerase chain reaction is a routinely used tool in every diagnostic and research laboratory. This technique has been used in detection of mutations and pathogens, forensic investigation, and even is the base tool for human genome sequencing. A modification of PCR technique, real time PCR, allows the quantification of nucleic acids with higher sensibility, specificity and reproducibility. This article is intended to clarify the foundations of real-time PCR, using an application model for virology. In the actual work, it was quantified the viral load of dengue virus serotype 2 produced from infected murine macrophages; the obtained results in this work established that murine strain BALB/c presents a greater susceptibility to dengue virus infection, which establishes BALB/c murine strain as a best model of study for investigation of dengue virus infection physiopathology.

  20. The Power of Real-Time PCR

    Science.gov (United States)

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

    In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene…

  1. [Usefulness of a real-time quantitative polymerase-chain reaction (PCR) assay for the diagnosis of congenital and postnatal cytomegalovirus infection].

    Science.gov (United States)

    Reina, J; Weber, I; Riera, E; Busquets, M; Morales, C

    2014-05-01

    Cytomegalovirus (CMV) is the main virus causing congenital and postnatal infections in the pediatric population. The aim of this study is to evaluate the usefulness of a quantitative real-time PCR in the diagnosis of these infections using urine as a single sample. We studied all the urine samples of newborns (PCR (PCRc), and quantitative real-time PCR (PCRq). We analyzed 332 urine samples (270 to rule out congenital infection and 62 postnatal infections). Of the first, 22 were positive in the PCRq, 19 in the PCRc, and 17 in the culture. PCRq had a sensitivity of 100%, on comparing the culture with the rest of the techniques. Using the PCRq as a reference method, culture had a sensitivity of 77.2%, and PCRc 86.3%. In cases of postnatal infection, PCRq detected 16 positive urines, the PCRq 12, and the cell culture 10. The urines showed viral loads ranging from 2,178 to 116,641 copies/ml. The genomic amplification technique PCRq in real time was more sensitive than the other techniques evaluated. This technique should be considered as a reference (gold standard), leaving the cell culture as a second diagnostic level. The low cost and the automation of PCRq would enable the screening for CMV infection in large neonatal and postnatal populations. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

  2. 实时荧光定量PCR技术研究进展及其应用%The Application and Development of Real-time Fluorescent Quantitative PCR

    Institute of Scientific and Technical Information of China (English)

    丁晓东; 马国文

    2006-01-01

    实时荧光定量PCR(Real Time Quantitative PCR)是把酶动力学、核酸扩增与杂交、光谱分析和实时检测技术巧妙结合的一项技术;并且广泛应用于病毒学、遗传性疾病、肿瘤和癌症的诊断等方面.因此成为分子生物学研究中的重要工具,本文就此技术研究进展及其应用做一综述.

  3. 实时荧光定量PCR技术原理与应用%Principles and Application of Real-time Fluorescent Quantitative PCR Technique

    Institute of Scientific and Technical Information of China (English)

    周晓丽; 朱国坡; 李雪华; 王艳玲; 刘兴友

    2010-01-01

    实时荧光定量PCR(real-time fluorescent quantitative polymerase chain reaction,FQ-PCR)是在定性PCR技术基础上发展起来的核酸定量技术,它具有特异性强、PCR污染少、自动化程度高等特点.作者就实时荧光定量PCR技术的主要原理及目前在分子生物学领域,病原体和肿瘤等方面的检测和诊断应用进行了概述.

  4. Real-time PCR in microfluidic devices

    Science.gov (United States)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.

    Science.gov (United States)

    Li, Xiuying; Yang, Qiwei; Bai, Jinping; Xuan, Yali; Wang, Yimin

    2015-01-01

    Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

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

    Science.gov (United States)

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

    2009-01-01

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

  8. 血清E型沙眼衣原体的Real-time PCR定量分析%Quantitative Analysis of Chlamydia trachomatis (serovar E) by Real-time PCR

    Institute of Scientific and Technical Information of China (English)

    李涛; 向志光; 林树柱; 秦川

    2011-01-01

    Objective Chlamydia trachomatis infection is the most common sexually transmitted disease. The aim of this research is to set a standard system for determination of chlamydia load in tissues from infected animals. Methods Chlamydia trachomatis (serovar E) was amplified in vitro for infection. The gene fragment of the chlamydia-specific gene OMP1 was cloned as standard. The quantity of chlamydia was determined based on the copy number of chlamydia genome by real time PCR. Results The results of real time PCR were linear when the copy number of OMP1 gene was within the range from 200 to 2 × 108. Unspecific amplification was not detected after adding mouse genome into the templates, nor was the amplification efficiency affected. Conclusion Real time PCR targeting chlamydia-specific gene OMP1 could be applied as a standard method to quantitatively analysis chlamydia in infected animal sample with adequate sensitivity and specificity.%目的 沙眼衣原体感染是最常见的性传播疾病,本文拟建立一种准确快速、标准化的感染动物组织衣原体载量检测体系.方法 体外扩增感染用沙眼衣原体血清E型,克隆衣原体特异基因OMP1基因片段作为标准品,用Real time PCR法测定衣原体基因组拷贝数进行衣原体定量.结果 Real time PCR在OMP1基因片段200至2×108拷贝检测结果成线性,在模板中加入小鼠基因组未出现非特异扩增,同时未影响扩增效率.结论 针对衣原体特异基因OMP1的实时定量PCR方法可以较为灵敏的特异的定量检测感染动物样本中的衣原体.

  9. Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients

    Directory of Open Access Journals (Sweden)

    De Vos Daniel

    2009-11-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols. Results In our hands, all three culture methods and the bioMérieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture. Conclusion In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.

  10. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD...... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...... axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer...

  11. Real-time PCR for quantitative analysis of human commensal Escherichia coli populations reveals a high frequency of subdominant phylogroups.

    Science.gov (United States)

    Smati, Mounira; Clermont, Olivier; Le Gal, Frédéric; Schichmanoff, Olivier; Jauréguy, Françoise; Eddi, Alain; Denamur, Erick; Picard, Bertrand

    2013-08-01

    Escherichia coli is divided into four main phylogenetic groups, which each exhibit ecological specialization. To understand the population structure of E. coli in its primary habitat, we directly assessed the relative proportions of these phylogroups from the stools of 100 healthy human subjects using a new real-time PCR method, which allows a large number of samples to be studied. The detection threshold for our technique was 0.1% of the E. coli population, i.e., 10(5) CFU/g of feces; in other methods based on individual colony analysis, the threshold is 10%. One, two, three, or four phylogenetic groups were simultaneously found in 21%, 48%, 21%, and 8% of the subjects, respectively. Phylogroups present at a threshold of less than 10% of the population were found in 40% of the subjects, revealing high within-individual diversity. Phylogroups A and B2 were detected in 74% and 70% of the subjects, respectively; phylogroups B1 and D were detected in 36% and 32%, respectively. When phylogroup B2 was dominant, it tended not to cooccur with other phylogroups. In contrast, other phylogroups were present when phylogroup A was dominant. These data indicate a complex pattern of interactions between the members of a single species within the human gut and identify a reservoir of clones that are present at a low frequency. The presence of these minor clones could explain the fluctuation in the composition of the E. coli microbiota within single individuals that may be seen over time. They could also constitute reservoirs of virulent and/or resistant strains.

  12. Development of a quantitative real-time PCR assay for detection of Vibrio tubiashii targeting the metalloprotease gene.

    Science.gov (United States)

    Gharaibeh, Dima N; Hasegawa, Hiroaki; Häse, Claudia C

    2009-03-01

    Vibrio tubiashii has recently re-emerged as a pathogen of bivalve larvae, causing a marked increase in the mortality of these species within shellfish rearing facilities. This has resulted in substantial losses of seed production and thus created the need for specific as well as sensitive detection methods for this pathogen. In this project, quantitative PCR (qPCR) primers were developed and optimized based upon analysis of the V. tubiashii vtpA gene sequence, encoding a metalloprotease known to cause larval mortality. Standard curves were developed utilizing dilutions of known quantities of V. tubiashii cells that were compared to colony forming unit (CFU) plate counts. The assay was optimized for detection of vtpA with both lab-grown V. tubiashii samples and filter-captured environmental seawater samples seeded with V. tubiashii. In addition, the primers were confirmed to specifically detect only V. tubiashii when tested against a variety of non-target Vibrio species. Validation of the assay was completed by analyzing samples obtained from a shellfish hatchery. The development of this rapid and sensitive assay for quantitative detection of V. tubiashii will accurately determine levels of this bacterium in a variety of seawater samples, providing a useful tool for oyster hatcheries and a method to assess the presence of this bacterium in the current turbulent ocean environment.

  13. Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

    Science.gov (United States)

    Brinkman, Nichole E.; Haugland, Richard A.; Wymer, Larry J.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Vesper, Stephen J.

    2003-01-01

    Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

  14. Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy.

    Directory of Open Access Journals (Sweden)

    María Martínez-Giner

    Full Text Available BACKGROUND: Reproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages. RESULTS: The gene expression stability of ten potential reference genes was studied by three different methods (geNorm, NormFinder and BestKeeper in ovary and uterus collected at five different physiological time points (heat, and 15, 30, 45 and 60 days of pregnancy. Although final ranking differed, the three algorithms gave very similar results. Thus, the most stable genes across time were TBP and UBC in uterus and TBP and HPRT1 in ovary, while HMBS and ACTB showed the less stable expression in uterus and ovary, respectively. When studied as a systematic effect, the reproductive stage did not significantly affect the expression of the candidate reference genes except at 30d and 60d of pregnancy, when a general drop in expression was observed in ovary. CONCLUSIONS: Based in our results, we propose the use of TBP, UBC and SDHA in uterus and TBP, GNB2L1 and HPRT1 in ovary for normalization of longitudinal expression studies using quantitative PCR in sows.

  15. Combining real-time PCR and next-generation DNA sequencing to provide quantitative comparisons of fungal aerosol populations

    Science.gov (United States)

    Dannemiller, Karen C.; Lang-Yona, Naama; Yamamoto, Naomichi; Rudich, Yinon; Peccia, Jordan

    2014-02-01

    We examined fungal communities associated with the PM10 mass of Rehovot, Israel outdoor air samples collected in the spring and fall seasons. Fungal communities were described by 454 pyrosequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal RNA encoding gene. To allow for a more quantitative comparison of fungal exposure in humans, the relative abundance values of specific taxa were transformed to absolute concentrations through multiplying these values by the sample's total fungal spore concentration (derived from universal fungal qPCR). Next, the sequencing-based absolute concentrations for Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, and Penicillium/Aspergillus spp. were compared to taxon-specific qPCR concentrations for A. alternata, C. cladosporioides, E. nigrum, and Penicillium/Aspergillus spp. derived from the same spring and fall aerosol samples. Results of these comparisons showed that the absolute concentration values generated from pyrosequencing were strongly associated with the concentration values derived from taxon-specific qPCR (for all four species, p 0.70). The correlation coefficients were greater for species present in higher concentrations. Our microbial aerosol population analyses demonstrated that fungal diversity (number of fungal operational taxonomic units) was higher in the spring compared to the fall (p = 0.02), and principal coordinate analysis showed distinct seasonal differences in taxa distribution (ANOSIM p = 0.004). Among genera containing allergenic and/or pathogenic species, the absolute concentrations of Alternaria, Aspergillus, Fusarium, and Cladosporium were greater in the fall, while Cryptococcus, Penicillium, and Ulocladium concentrations were greater in the spring. The transformation of pyrosequencing fungal population relative abundance data to absolute concentrations can improve next-generation DNA sequencing-based quantitative aerosol exposure assessment.

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

    Science.gov (United States)

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

    2014-08-01

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

  17. Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

    Directory of Open Access Journals (Sweden)

    Pier Luigi Acutis

    2011-11-01

    Full Text Available The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci. An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells.

  18. Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

    Science.gov (United States)

    Peletto, Simone; Bertuzzi, Simone; Campanella, Chiara; Modesto, Paola; Maniaci, Maria Grazia; Bellino, Claudio; Ariello, Dario; Quasso, Antonio; Caramelli, Maria; Acutis, Pier Luigi

    2011-01-01

    The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells. PMID:22174628

  19. Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy.

    Science.gov (United States)

    Bergallo, Massimiliano; Galliano, Ilaria; Montanari, Paola; Brusin, Martina Rosa; Finotti, Serena; Paderi, Giulia; Gabiano, Clara

    2016-12-02

    Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log10) ± 7.1(log10) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

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

    NARCIS (Netherlands)

    Dijksman, Johan Frederik; Pierik, A.

    2012-01-01

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

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

    NARCIS (Netherlands)

    Dijksman, J.F.; Pierik, A.

    2012-01-01

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

  2. Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR.

    Science.gov (United States)

    El-Sherbini, Yasser M; Stevenson, Mark M; Seymour, Leonard W; Wade-Martins, Richard

    2009-08-01

    Herpes simplex virus type 1 (HSV-1) amplicon preparations are usually quantified as transducing units/ml (TU/ml), with little information on genomic copy/TU ratios. In the present study, two HSV-1 amplicons expressing enhanced green fluorescent protein (EGFP) were analysed by quantitative PCR (qPCR) and transducing activity to obtain genomic copy/TU ratios. One vector (pHSV-GL) contains the HSV-1 packaging signal (pac) and origin of replication (oriS) and the other (pHSV/EBV-GL) includes Epstein-Barr virus (EBV) episomal maintenance elements. The pHSV-GL and pHSV/EBV-GL amplicons were prepared at titres of 7.55x10(7) and 7.24x10(7)TU/ml, containing 2.56x10(9) and 1.33x10(9) genomic copies/ml respectively. This produced preliminary estimates of genomic copy/TU ratios of 34:1 and 18:1. However standard transduction conditions did not deplete fully the supernatant of transducing particles since the same supernatant was subsequently able to achieve 25% the initial transduction efficiency, although centrifugation of amplicon particles onto cells improved infectivity by 1.8-fold. Finally, qPCR analysis of FACS-purified EGFP-expressing cells showed the presence of approximately 3 amplicon genomes/transduced cell, independent of the infection dose. Accordingly, the initial estimated genomic copy/TU ratio for pHSV-GL was revised to 6.3:1. Measuring the genomic copy/TU ratios is an important parameter for comparing the quality of amplicon preparations and standardizing experimental conditions.

  3. Prognostic impact of tumour burden measured by quantitative real-time PCR from sentinel lymph nodes of melanoma patients: data from 10-year follow-up.

    Science.gov (United States)

    Eigentler, Thomas Kurt; Hinderer, Joachim; Noor, Seema; Garbe, Claus; Leiter, Ulrike

    2017-04-01

    Sentinel node (SN) biopsy is regarded as standard of care for patients (pts) with cutaneous melanoma ≥1.0 mm of thickness. In the recent AJCC classification, findings in the SN are simply classified as positive or negative. In our analyses, we were interested whether quantitative real-time PCR (qRT-PCR) is able to predict disease-free survival (DFS) and overall survival (OS) depending on tumour burden in the SN. One hundred and forty-five pts were analysed using qRT-PCR for tyrosinase. Results were analysed using accelerated failure time survival model and cox proportional hazards models using the R statistics framework. Forty-one pts (28%) were positive according to qRT-PCR. In total, 12 of 41 pts showed tumour deposits in the SN using S100 and/or HMB-45-labelled immunohistochemistry as well. One patient had micrometastases detected by immunohistochemistry staining but failed in the qRT-PCR. After 10 years of follow-up, 34 patients recurred and 27 patients died. Significant differences for DFS and OS were detected for sex, increasing tumour thickness, ulceration of the primary tumour, and metastatic spread in the SN determined by histology as well as qRT-PCR. Quantitative analyses showed a logarithmic correlation between tumour burden and prognosis. However, as multivariate analyses reveal qRT-PCR was not superior compared to classical histology or immunohistology.

  4. A comparison of real-time PCR protocols for the quantitative monitoring of asymptomatic olive infections by Verticillium dahliae pathotypes.

    Science.gov (United States)

    Gramaje, D; Pérez-Serrano, V; Montes-Borrego, M; Navas-Cortés, J A; Jiménez-Díaz, R M; Landa, B B

    2013-10-01

    Early, specific, and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity, and efficiency of eight real-time quantitative polymerase chain reaction protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems, and leaves. Results showed that some of those protocols were not specific for V. dahliae or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocols TAQ (based on intergenic spacer ribosomal DNA target gene) and SYBR-4 (based on the β-tubulin 2 target gene) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and stem) tested. Use of TAQ and SYBR-4 protocols allowed accurate quantification of V. dahliae DNA regardless of the background DNA, with a detection limit being fixed at a cycle threshold of 36 (≈18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae. The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant 'Frantoio' olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D V. dahliae DNA were measured in olive stems, whereas the average amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amounts in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days after inoculation). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing

  5. Allele frequency of hereditary equine regional dermal asthenia in American Quarter horses in Brazil determined by quantitative real-time PCR with high resolution melting analysis.

    Science.gov (United States)

    Badial, Peres R; Oliveira-Filho, José P; Winand, Nena J; Borges, Alexandre S

    2014-02-01

    Hereditary equine regional dermal asthenia (HERDA) is a genetic disorder that occurs in the American Quarter horse (AQH) and is caused by a c.115G>A missense mutation in the peptidylprolyl isomerase B (PPIB) gene. Using a quantitative real-time PCR high resolution melting analysis genotyping assay for the PPIB mutation, the estimated HERDA allele and carrier frequencies in a sample of Brazilian AQHs were 2.9% and 5.8%, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotype of F. graminearum and F. culmorum isolates in Danish small grain cereals

    DEFF Research Database (Denmark)

    Nielsen, L. K.; Jensen, J. D.; Rodríguez, A.

    2012-01-01

    Quantitative real-time PCR assays, based on polymorphisms in the TRI12 gene of the trichothecene pathway, were developed to identify and quantify the trichothecene genotypes producing 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) or nivalenol (NIV) in the Fusarium graminearum...... in the sample representing the years from 1997 to 2000. Detection of low amounts of the 15ADON genotype in these historical samples and the relatively high amounts of 15ADON genotype in 2003 and following years correspond well with the occurrence of F. graminearum and indicates that the 15ADON genotype...

  7. Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer

    DEFF Research Database (Denmark)

    Reis, Patricia P; Rogatto, Silvia R; Kowalski, Luiz P;

    2002-01-01

    of disease (P=0.025), family history of cancer (P=0.001), and death (P=0.021). Relative copy number loss involving the DIA1 gene was correlated to family history of cancer (Palcohol (P=0.026). Log-rank test revealed a significant decrease in survival (P=0......Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity...... findings in head and neck tumors. Quantitative PCR relies on the comparison of the amount of product generated from a target gene and that generated from a disomic reference gene (GAPDH-housekeeping gene). Reactions have been performed with normal control in triplicates, using the 7700 Sequence Detection...

  8. Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR

    DEFF Research Database (Denmark)

    Wang, Chong; Robles, Francisco; Ramirez, Saul

    2016-01-01

    Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real-time quant...

  9. Factor optimization for the reaction of real-time fluorescent quantitative PCR%实时荧光定量PCR反应体系的优化研究

    Institute of Scientific and Technical Information of China (English)

    袁坤; 张丹; 刘新琼; 张向明

    2013-01-01

    为了降低实时荧光定量PCR(Real-Time Fluorescent Quantitative PCR,FQ-PCR)技术的实验成本,提高利用效率,对影响FQ-PCR较敏感的因素二甲基亚砜(DMSO)、甘油和Mg2+等进行了优化试验.结果表明,10μL反应体系的最优组分为:1×rTaqBuffer,0.16 mmol/L dNTPs,0.24μmol/L Primer,2.5 mmol/L Mg2+,5%甘油,5% DMSO,1×SYBR Green I,0.8 U rTaq酶,100 ng模板.该反应体系为经济型FQ-PCR的广泛应用奠定了基础.

  10. Targeted profiling of oral bacteria in human saliva and in vitro biofilms with quantitative real-time PCR.

    Science.gov (United States)

    Price, R R; Viscount, H B; Stanley, M C; Leung, K-P

    2007-01-01

    An in vitro plaque model based on the use of human salivary bacteria and tooth-like surfaces was previously developed for studying the formation of oral biofilm and its use for pre-clinical testing of candidate antimicrobial or antiplaque agents. In this study, a quantitative Taqman PCR assay (QPCR) was developed to compare the bacterial compositions of in vitro biofilms to parent saliva samples, and to determine the relative contributions of different species in the formation of the oral biofilm. In addition, the growth inhibition of saliva-derived plaque was evaluated by chlorhexidine. With this assay, which consisted of primer/probe sets targeting either 16S rDNA sequences present in public databases or cloned ribosomal intergenic spacer region (ISR) sequences, 15 oral bacteria derived from saliva as well as those that were responsible for biofilm formation in an in vitro plaque model were rapidly identified and quantified. Among the target organisms were Actinobacillus actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Lactobacillus acidophilus, Micromonas micros, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Streptococcus sobrinus, Tannerella forsythensis, and Veillonella parvula. Primer and probe sets developed were both sensitive and specific. The relative profiles of a number of bacteria in 45-h-old biofilms were determined and, when compared to saliva samples, it was found that most of the bacteria identified in saliva also populated the in vitro plaque, including some anaerobes. Brief exposure of biofilms to chlorhexidine resulted in significant losses in viability. This new broad spectrum QPCR assay in combination with the in vitro plaque model will be of significant value in the quantitative study of the microbial composition of human saliva, saliva-derived plaque, and pre-clinical evaluation of potential antimicrobial and antiplaque molecules.

  11. Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

    Institute of Scientific and Technical Information of China (English)

    Qiu You-wen; Gao Xue-jun; Qi Bang-ruo; Li Lu; Zhen Zhen

    2012-01-01

    TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

  12. Species-specific identification of ruminant components contaminating industrial crude porcine heparin using real-time fluorescent qualitative and quantitative PCR.

    Science.gov (United States)

    Huang, Qing; Xu, Ting; Wang, Gui-Yu; Huang, Jun-Fu; Xia, Han; Yin, Richard; Tang, Angie; Fu, Wei-Ling

    2012-02-01

    Ever since the emergence of bovine spongiform encephalopathy, the source of pharmaceutical heparin has been restricted to porcine intestinal mucosa. In this project, two real-time fluorescent PCR methods were developed to assist with quality control analysis. The first is a qualitative method which relies on SYBR Green I chemistry to confirm the porcine origin of industrial crude porcine heparin (ICPH), identify any ruminant contaminants, and generally control purity. The second is based on TaqMan chemistry and is able to quantitatively identify porcine, bovine, caprine, and ovine components and contaminants in ICPH. By targeting mitochondrial DNA, both PCR systems showed a detection limit of 1 pg DNA and amplification efficiencies ranging between 96% and 102%. Moreover, quantitative PCR showed a detection limit of 0.02 ppm in samples comprising porcine, bovine, caprine, and ovine DNA. The results of qualitative PCR over 27 ICPH samples showed that all samples were porcine in origin and that 17 had ruminant contaminants. The results of quantitative PCR further showed that out of all 17 samples with ruminant contaminants, seven samples had bovine, ovine, and caprine contaminants, two samples had bovine and ovine contaminants, and eight samples had only ovine contaminants. In conclusion, the qualitative PCR system was found to be a relatively inexpensive, rapid, and flexible method of identifying the porcine origin of and ruminant contaminants in ICPH, while the quantitative PCR was found suitable to accurately analyze the components and contaminants in detail. Both methods are suitable for routine control assays for the evaluation of ICPH purity and origins of contaminants.

  13. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    Science.gov (United States)

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  14. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

    Science.gov (United States)

    Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

    2016-07-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  15. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

    Science.gov (United States)

    Fu, Hua-Ying; Sun, Sheng-Ren; Wang, Jin-Da; Ahmad, Kashif; Wang, Heng-Bo; Chen, Ru-Kai

    2016-01-01

    Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  16. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

    Directory of Open Access Journals (Sweden)

    Hua-Ying Fu

    2016-01-01

    Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  17. Clinical application of real-time PCR to screening critically ill and emergency-care surgical patients for methicillin-resistant Staphylococcus aureus: a quantitative analytical study.

    Science.gov (United States)

    Herdman, M Trent; Wyncoll, Duncan; Halligan, Eugene; Cliff, Penelope R; French, Gary; Edgeworth, Jonathan D

    2009-12-01

    The clinical utility of real-time PCR screening assays for methicillin (methicillin)-resistant Staphylococcus aureus (MRSA) colonization is constrained by the predictive values of their results: as MRSA prevalence falls, the assay's positive predictive value (PPV) drops, and a rising proportion of positive PCR assays will not be confirmed by culture. We provide a quantitative analysis of universal PCR screening of critical care and emergency surgical patients using the BD GeneOhm MRSA PCR system, involving 3,294 assays over six months. A total of 248 PCR assays (7.7%) were positive; however, 88 failed to be confirmed by culture, giving a PPV of 65%. Multivariate analysis was performed to compare PCR-positive culture-positive (P+C+) and PCR-positive culture-negative (P+C-) assays. P+C- results were positively associated with a history of methicillin-sensitive Staphylococcus aureus infection or colonization (odds ratio [OR], 3.15; 95% confidence interval [CI], 1.32 to 7.54) and high PCR thresholds of signal intensity, indicative of a low concentration of target DNA (OR, 1.19 per cycle; 95% CI, 1.11 to 1.26). P+C- results were negatively associated with a history of MRSA infection or colonization (OR, 0.19; 95% CI, 0.09 to 0.42) and male sex (OR, 0.40; 95% CI, 0.20 to 0.81). P+C+ patients were significantly more likely to have subsequent positive MRSA culture assays and microbiological evidence of clinical MRSA infection. The risk of subsequent MRSA infection in P+C- patients was not significantly different from that in case-matched PCR-negative controls. We conclude that, given the low PPV and poor correlation between a PCR-positive assay and the clinical outcome, it would be prudent to await culture confirmation before altering infection control measures on the basis of a positive PCR result.

  18. Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2003-01-01

    Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

  19. Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR

    Institute of Scientific and Technical Information of China (English)

    LI Bing; CHEN Changsheng; XU Yan; JI Dehua; XIE Chaotian

    2014-01-01

    Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expres-sion studies using quantitative real-time PCR should start by selecting an appropriate internal control gene;therefore, the absolute expression abundance of six housekeeping genes (18S rRNA (18S), ubiquitin-conju-gating enzyme (UBC), actin (ACT),β-tubulin (TUB), elongation factors 2 (EF2), and glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold (Ct) method and by two different software packages:geNorm and Norm-Finder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.

  20. Quantitation of HTLV-I proviral load by a real-time PCR assay using SYBR Green: comparison of two methods for DNA isolation.

    Science.gov (United States)

    Altamirano, Natalia Andrea; Rocco, Carlos; Aulicino, Paula; Sen, Luisa; Mangano, Andrea

    2010-12-01

    A real-time quantitative PCR (qPCR) assay using SYBR Green was developed to determine HTLV-I proviral load (pVL) in peripheral blood mononuclear cells (PBMCs), and its performance was evaluated with samples processed as cell lysates and DNA isolated by salting out. Primers targeting the pol region were standardized against the MT2 cell line and HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the albumin gene. The sensitivity, specificity and reproducibility of the qPCR were assessed in the two methods used for DNA processing. The assay had a limit of detection of 400 HTLV-I copies/10(6) PBMCs for both methods, with a broad range of quantitation (2.6log(10) to >5log(10)), and without cross-reactivity with HTLV-II or with HIV-1. The inter- and intra-assay coefficients of variation were less than 2.4%. HTLV-I pVL quantitation in seven blood donor samples processed as either cell lysates or isolated DNA by salting out showed a strong linear correlation and no difference in the calculated pVL (Fisher's exact test, p>0.05). The assay was found to be a low cost, robust and reproducible assay for quantifying HTLV-I pVL in samples processed as cell lysates or as isolated DNA.

  1. Application of SSH and quantitative real time PCR to construction of gene expression profiles from scallop Chlamys farreri in response to exposure to tetrabromobisphenol A.

    Science.gov (United States)

    Gong, Xiaoli; Pan, Luqing; Miao, Jingjing; Liu, Na

    2012-11-01

    TBBPA-induced genes were identified using suppression subtractive hybridization (SSH) from Chlamys farreri. A total of 203 and 44 clones from SSH forward and reverse library were respectively obtained including cellular process, immune system process, response to stimulus, metabolic process and signaling etc. Differential gene expressions were compared between scallops from control and TBBPA treatment groups (400 μg/L, 15 days) using quantitative real time RT-PCR. For further research, eight significant genes expression from scallops exposed to TBBPA (0; 100; 200; 400 μg/L) sampling at 0, 1, 3, 6 and 15 days, were utilized for Q-RT-PCR. The results revealed that the expression level of most selected cDNAs was dominantly up-regulated or down-regulated in the TBBPA-induced scallops. These findings provide basic genomic information of the bivalve and the selected genes may be the potential molecular biomarkers for TBBPA pollution in aquatic environment.

  2. Galactomannan enzyme immunoassay and quantitative Real Time PCR as tools to evaluate the exposure and response in a rat model of aspergillosis after posaconazole prophylaxis.

    Science.gov (United States)

    Cendejas-Bueno, Emilio; Forastiero, Agustina; Ruiz, Isabel; Mellado, Emilia; Buitrago, María José; Gavaldà, Joan; Gomez-Lopez, Alicia

    2016-11-01

    A steroid-immunosuppressed rat model of invasive pulmonary aspergillosis was use to examine the usefulness of galactomannan enzyme immunoassay (GM) and quantitative real time PCR (RT-PCR) in evaluating the association between response and exposure after a high dose of prophylactic posaconazole. Two different strains of Aspergillus fumigatus with different in vitro posaconazole susceptibility were used. Serum concentrations demonstrated similar posaconazole exposure for all treated animals. However, response to posaconazole relied on the in vitro susceptibility of the infecting strain. After prophylaxis, galactomannan index and fungal burden only decreased in those animals infected with the most susceptible strain. This study demonstrated that both biomarkers may be useful tools for predicting efficacy of antifungal compounds in prophylaxis. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  3. Real-time quantitative PCR assay with Taqman® probe for rapid detection of MCR-1 plasmid-mediated colistin resistance

    Directory of Open Access Journals (Sweden)

    S. Chabou

    2016-09-01

    Full Text Available Here we report the development of two rapid real-time quantitative PCR assays with TaqMan® probes to detect the MCR-1 plasmid-mediated colistin resistance gene from bacterial isolates and faecal samples from chickens. Specificity and sensitivity of the assay were 100% on bacterial isolates including 18 colistin-resistant isolates carrying the mcr-1 gene (six Klebsiella pneumoniae and 12 Escherichia coli with a calibration curve that was linear from 101 to 108 DNA copies. Five out of 833 faecal samples from chickens from Algeria were positive, from which three E. coli strains were isolated and confirmed to harbour the mcr-1 gene by standard PCR and sequencing.

  4. Enumeration of Salmonellae in Table Eggs, Pasteurized Egg Products, and Egg-Containing Dishes by Using Quantitative Real-Time PCR

    DEFF Research Database (Denmark)

    Jakočiūnė, Džiuginta; Pasquali, Frédérique; da Silva, Cristiana Soares

    2014-01-01

    PCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between......Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (q...... the numbers of salmonellae and the quantification cycle (Cq) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate...

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

    NARCIS (Netherlands)

    Dijksman J.F.; Pierik, A.

    2012-01-01

    Real Time Array PCR is a recently developed biochemical technique that measures amplification curves (like quantitative real time Polymerase Chain Reaction (qPCR)) of a multitude of different templates ina sample. It combines two different techniques to profit from theadvantages of both techniques,

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

    NARCIS (Netherlands)

    Dijksman J.F.; Pierik, A.

    2012-01-01

    Real Time Array PCR is a recently developed biochemical technique that measures amplification curves (like quantitative real time Polymerase Chain Reaction (qPCR)) of a multitude of different templates ina sample. It combines two different techniques to profit from theadvantages of both techniques,

  7. Comprehensive selection of reference genes for expression studies in meniscus injury using quantitative real-time PCR.

    Science.gov (United States)

    Leal, Mariana Ferreira; Arliani, Gustavo Gonçalves; Astur, Diego Costa; Franciozi, Carlos Eduardo; Debieux, Pedro; Andreoli, Carlos Vicente; Smith, Marília Cardoso; Pochini, Alberto de Castro; Ejnisman, Benno; Cohen, Moises

    2016-06-10

    The meniscus plays critical roles in the knee function. Meniscal tears can lead to knee osteoarthritis. Gene expression analysis may be a useful tool for understanding meniscus tears, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. We evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1 and TBP) using meniscus samples of (1) 19 patients with isolated meniscal tears, (2) 20 patients with meniscal tears and combined anterior cruciate ligament injury (ACL), and (3) 11 controls without meniscal tears. The stability of the candidate reference genes was determined using the NormFinder, geNorm, BestKeeper DataAssist and RefFinder software packages and comparative ΔCt method. Overall, HPRT1 was the best single reference gene. However, GenEx software demonstrated that two or more reference genes should be used for gene expression normalization, which was confirmed when we evaluated TGFβR1 expression using several reference gene combinations. HPRT1+TBP was the most frequently identified pair from the analysis of samples of (1) meniscal tear samples of patients with a concomitant ACL tears, (2) all meniscal tears, and (3) all samples. HPRT1+GAPDH was the most frequently identified pair from the analysis of samples of isolated meniscal tear samples and controls. In the analysis involving only controls, GAPDH+18S was the most frequently identified pair. In the analysis of only isolated meniscal tear samples and in the analysis of meniscal tear samples of patients with concomitant ACL tears and controls, both HPRT1+TBP and HPRT1+GAPDH were identified as suitable pairs. If the gene expression study aims to compare non-injured meniscus, isolated meniscal tears and meniscal tears of patients with ACL tears as three independent groups, the trio of HPRT1+TBP+GAPDH is the most suitable

  8. Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

    OpenAIRE

    Pier Luigi Acutis; Maria Caramelli; Antonio Quasso; Dario Ariello; Claudio Bellino; Maria Grazia Maniaci; Paola Modesto; Chiara Campanella; Simone Bertuzzi; Simone Peletto

    2011-01-01

    The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes b...

  9. Real-time PCR in Food Science: Introduction.

    Science.gov (United States)

    Rodriguez-Lazaro, David; Hernandez, Marta

    2013-01-01

    Food safety and quality control programmes are increasingly applied throughout the production food chain in order to guarantee added value products as well as to minimize the risk of infection for the consumer. The development of real-time PCR has represented one of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly important for the agricultural and food industry. Different strategies for real-time PCR diagnostics have been developed including unspecific detection independent of the target sequence using fluorescent dyes such as SYBR Green, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

  10. Low incidence of minor BRAF V600 mutation-positive subclones in primary and metastatic melanoma determined by sensitive and quantitative real-time PCR

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Clemmensen, Ole; Hoejberg, Lise

    2013-01-01

    BRAF V600 mutation is an important biological marker for therapeutic guidance in melanoma, where mutation-positive cases are candidates for therapy targeting mutant B-Raf. Recent studies showing intratumor variation in BRAF mutation status have caused concern that sensitive mutation analysis can...... lead to mutation-positive results in patients with melanomas with small subsets of mutation-positive cells who may not benefit from therapy targeting mutant B-Raf. Mutation analysis with high analytical sensitivity is generally preferred, to reduce the risk of false-negative results. In this study......, sensitive and quantitative BRAF V600E and V600K mutation-specific real-time quantitative PCR was used to study the occurrence of small subsets of mutation-positive cells in primary melanomas and melanoma metastases. The BRAF V600E mutation was detected in 39 of 82 melanoma patients. We observed a highly...

  11. Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR.

    Science.gov (United States)

    Wang, Juan; Zhao, Shasha; Zhou, Ying; Wei, Yun; Deng, Wensheng

    2015-01-01

    Primer extension-dependent in vitro transcription assay is one of the most important approaches in the research field of gene transcription. However, conventional in vitro transcription assays incorporates radioactive isotopes that cause environmental and health concerns and restricts its scope of application. Here we report a novel non-radioactive method for in vitro transcription analysis by combining primer extension with quantitative real time PCR (qPCR). We show that the DNA template within the transcription system can be effectively eliminated to a very low level by our specially designed approach, and that the primers uniquely designed for primer extension and qPCR can specifically recognize the RNA transcripts. Quantitative PCR data demonstrate that the novel method has successfully been applied to in vitro transcription analyses using the adenovirus E4 and major late promoters. Furthermore, we show that the TFIIB recognition element inhibits transcription of TATA-less promoters using both conventional and nonradioactive in vitro transcription assays. Our method will benefit the laboratories that need to perform in vitro transcription but either lack of or choose to avoid radioactive facilities.

  12. Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment.

    Science.gov (United States)

    Josefsen, M H; Löfström, C; Hansen, T B; Christensen, L S; Olsen, J E; Hoorfar, J

    2010-08-01

    A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (C(T)) values (R(2) = 0.993), with a quantification range of 1 x 10(2) to 1 x 10(7) CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R(2) = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.

  13. Simultaneous Quantification and Differentiation of Streptococcus suis Serotypes 2 and 9 by Quantitative Real-Time PCR, Evaluated in Tonsillar and Nasal Samples of Pigs

    Directory of Open Access Journals (Sweden)

    Niels Dekker

    2016-06-01

    Full Text Available Invasive Streptococcus suis (S. suis infections in pigs are often associated with serotypes 2 and 9. Mucosal sites of healthy pigs can be colonized with these serotypes, often multiple serotypes per pig. To unravel the contribution of these serotypes in pathogenesis and epidemiology, simultaneous quantification of serotypes is needed. A quantitative real-time PCR (qPCR targeting cps2J (serotypes 2 and 1/2 and cps9H (serotype 9 was evaluated with nasal and tonsillar samples from S. suis exposed pigs. qPCR specifically detected serotypes in all pig samples. The serotypes loads in pig samples estimated by qPCR showed, except for serotype 9 in tonsillar samples (correlation coefficient = 0.25, moderate to strong correlation with loads detected by culture (correlation coefficient > 0.65, and also in pigs exposed to both serotypes (correlation coefficient > 0.75. This qPCR is suitable for simultaneous differentiation and quantification of important S. suis serotypes.

  14. Quantification of microcystin-producing and non-microcystin producing Microcystis populations during the 2009 and 2010 blooms in Lake Taihu using quantitative real-time PCR

    Institute of Scientific and Technical Information of China (English)

    Daming Li; Fanxiang Kong; Xiaoli Shi; Linlin Ye; Yang Yu; Zhen Yang

    2012-01-01

    Lake Taihu,a large,shallow hypertrophic freshwater lake in eastern China,has experienced lake-wide toxic cyanobacterial blooms annually during summer season in the past decades.Spatial changes in the abundance of hepatotoxin microcystin-producing and nonmicrocystin producing Microcystis populations were investigated in the lake in August of 2009 and 2010.To monitor the densities of the total Microcystis population and the potential microcystin-producing subpopulation,we used a quantitative real-time PCR assay targeting the phycocyanin intergenic spacer (PC-IGS) and the microcystin synthetase gene (mcyD),respectively.On the basis of quantification by real-time PCR analysis,the abundance of potential toxic Microcystis genotypes and the ratio of the mcyD subpopulation to the total Microcystis varied significantly,from 4.08×104 to 5.22×107 copies/mL,from 5.7% to 65.8%,respectively.Correlation analysis showed a strong positive relationship between chlorophyll-a,toxic Microcystis and total Microcystis; the abundance of toxic Microcystis correlated positively with total phosphorus and ortho-phosphate concentrations,but negatively with TN:TP ratio and nitrate concentrations.Meanwhile the proportion of potential toxic genotypes within Microcystis population showed positive correlation with total phosphorus and ortho-phosphate concentrations.Our data suggest that increased phosphorus loading may be a significant factor promoting the occurrence of toxic Microcystis bloom in Lake Taihu.

  15. Rapid detection of Streptococcus pneumoniae by real-time fluorescence quantitation PCR%实时荧光定量PCR快速诊断肺炎链球菌

    Institute of Scientific and Technical Information of China (English)

    颜善活; 孙雷; 符可鹏; 卓永光; 杨善业; 樊祖茜; 黄永霞

    2013-01-01

    目的 建立实时荧光定量聚合酶链反应(PCR),用于肺炎链球菌检测和流行病学调查.方法 以肺炎链球菌自溶素(lyt)和溶血素(ply)的基因为目的序列分别设计引物和探针,将其分别与10株肺炎链球菌、13株非肺炎链球菌DNA以及不同浓度梯度的肺炎链球菌DNA进行荧光定量PCR,并将引物和探针应用于200例临床标本检测,同时通过传统的微生物培养鉴定的方法来验证荧光定量PCR的特异性和敏感性.结果 10株肺炎链球菌均获得了明显的扩增产物,13株非肺炎链球菌DNA无明显的扩增信号,其检测敏感性可达100 fg;200例临床标本,实时荧光定量PCR检测出42例肺炎链球菌阳性(阳性率为21%),而培养法阳性16例(阳性率为8%).结论 实时荧光定量PCR是一种敏感、特异、快速的检测肺炎链球菌方法,可用于肺炎链球菌的诊断和流行病学调查.%Objective To establish real-time fluorescence quantitation polymerase chain reaction ( PCR) for the detection and epidemiological studies of Streptococcus pneumoniae. Methods The autolysin (lyt) gene and hemolysin (ply) gene sequences were selected to develop primers and probe. A total of 10 strains of Streptococcus pneumoniae,13 strains of non-Streptococcus pneumoniae DISA and the different concentration strains of Streptococcus pneumoniae DNA were detected by real-time fluorescence quantitation PCR, and 200 clinical specimens were detected by primers and probe. The specificity and sensitivity were also analyzed. Results The 10 strains of Streptococcus pneumoniae were measured to obtain amplification products. However, 13 strains of non-Streptococcus pneumoniae DNA had no obvious amplification, and the sensitivity was 100fg. Among the 200 clinical specimens, real-time fluorescence quantitation PCR detected 42 cases of Streptococcus pneumoniae-positive (the positive rate was 21% ), while the culture method detected 16 cases of Streptococcus pneumoniae

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

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2016-01-01

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

  17. Determination of Cytochrome P450 2D6 (CYP2D6 Gene Copy Number by Real-Time Quantitative PCR

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    Laurent Bodin

    2005-01-01

    Full Text Available Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number. Using the 2−ΔΔCt calculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies, and 12 samples with duplicated genes. The average ratio ranged from 1.02 to 1.28, 1.85 to 2.21, and 2.55 to 3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication.

  18. New in situ capture quantitative (real-time) reverse transcription-PCR method as an alternative approach for determining inactivation of Tulane virus.

    Science.gov (United States)

    Wang, Dapeng; Xu, Shuxia; Yang, David; Young, Glenn M; Tian, Peng

    2014-04-01

    Human noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method. We employed type B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome by in situ qRT-PCR. We first demonstrated that this in situ capture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by the ISC-qRT-PCR method and confirmed by TCID50 assay. However, the loss of the infectivity caused by damage to the viral genome (such as that from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively

  19. Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station.

    Science.gov (United States)

    Parra, Macarena; Jung, Jimmy; Boone, Travis D; Tran, Luan; Blaber, Elizabeth A; Brown, Mark; Chin, Matthew; Chinn, Tori; Cohen, Jacob; Doebler, Robert; Hoang, Dzung; Hyde, Elizabeth; Lera, Matthew; Luzod, Louie T; Mallinson, Mark; Marcu, Oana; Mohamedaly, Youssef; Ricco, Antonio J; Rubins, Kathleen; Sgarlato, Gregory D; Talavera, Rafael O; Tong, Peter; Uribe, Eddie; Williams, Jeffrey; Wu, Diana; Yousuf, Rukhsana; Richey, Charles S; Schonfeld, Julie; Almeida, Eduardo A C

    2017-01-01

    The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Identification and Evaluation of Suitable Reference Genes for Gene Expression Studies in the Whitefly Bemisia tabaci (Asia I) by Reverse Transcription Quantitative Real-Time PCR

    Science.gov (United States)

    Collins, Carl; Patel, Mitulkumar V.; Colvin, John; Bailey, David; Seal, Susan

    2014-01-01

    This study presents a reliable method for performing reverse transcription quantitative real-time PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data presented here will assist future transcript profiling studies in whiteflies. PMID:25373210

  2. Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells.

    Science.gov (United States)

    Li, X; Huang, K; Chen, F; Li, W; Sun, S; Shi, X-E; Yang, G

    2016-06-01

    Intramuscular fat (IMF) is an important trait influencing meat quality, and intramuscular stromal-vascular cell (MSVC) differentiation is a key factor affecting IMF deposition. Quantitative real-time PCR (qPCR) is often used to screen the differentially expressed genes during differentiation of MSVCs, where proper reference genes are essential. In this study, we assessed 31 of previously reported reference genes for their expression suitability in porcine MSVCs derived form longissimus dorsi with qPCR. The expression stability of these genes was evaluated using NormFinder, geNorm and BestKeeper algorithms. NormFinder and geNorm uncovered ACTB, ALDOA and RPS18 as the most three stable genes. BestKeeper identified RPL13A, SSU72 and DAK as the most three stable genes. GAPDH was found to be the least stable gene by all of the three software packages, indicating it is not an appropriate reference gene in qPCR assay. These results might be helpful for further studies in pigs that explore the molecular mechanism underlying IMF deposition.

  3. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea in soil and fecal samples

    Directory of Open Access Journals (Sweden)

    Durant Jean-Francois

    2012-12-01

    Full Text Available Abstract Background Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis and/or Toxocara cati (T. cati, two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. Methods A duplex quantitative real-time PCR (2qPCR targeting the ribosomal RNA gene internal transcribed spacer (ITS2 has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum and Parascaris equorum (P. equorum. The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. Results 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. Conclusion The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

  4. Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR

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    Longjian Niu

    2015-06-01

    Full Text Available Real-time quantitative PCR (RT-qPCR is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis, a promising oilseed crop known for its polyunsaturated fatty acid (PUFA-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt, BestKeeper, geNorm, and NormFinder were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE, actin (ACT and phospholipase A22 (PLA were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13, cyclophilin (CYC and elongation factor-1alpha (EF1α were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

  5. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  6. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  7. Identification of endogenous control genes for normalisation of real-time quantitative PCR data in colorectal cancer.

    LENUS (Irish Health Repository)

    Kheirelseid, Elrasheid A H

    2010-01-01

    BACKGROUND: Gene expression analysis has many applications in cancer diagnosis, prognosis and therapeutic care. Relative quantification is the most widely adopted approach whereby quantification of gene expression is normalised relative to an endogenously expressed control (EC) gene. Central to the reliable determination of gene expression is the choice of control gene. The purpose of this study was to evaluate a panel of candidate EC genes from which to identify the most stably expressed gene(s) to normalise RQ-PCR data derived from primary colorectal cancer tissue. RESULTS: The expression of thirteen candidate EC genes: B2M, HPRT, GAPDH, ACTB, PPIA, HCRT, SLC25A23, DTX3, APOC4, RTDR1, KRTAP12-3, CHRNB4 and MRPL19 were analysed in a cohort of 64 colorectal tumours and tumour associated normal specimens. CXCL12, FABP1, MUC2 and PDCD4 genes were chosen as target genes against which a comparison of the effect of each EC gene on gene expression could be determined. Data analysis using descriptive statistics, geNorm, NormFinder and qBasePlus indicated significant difference in variances between candidate EC genes. We determined that two genes were required for optimal normalisation and identified B2M and PPIA as the most stably expressed and reliable EC genes. CONCLUSION: This study identified that the combination of two EC genes (B2M and PPIA) more accurately normalised RQ-PCR data in colorectal tissue. Although these control genes might not be optimal for use in other cancer studies, the approach described herein could serve as a template for the identification of valid ECs in other cancer types.

  8. DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS

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    M. A. Gordukova

    2015-01-01

    Full Text Available Primary immunodeficiencies (PID such as severe combined immunodeficiency (SCID and X-linked agammaglobulinemia are characterized by the lack of functional Tand B-cells, respectively. Without early diagnosis and prompt treatment children with PID suffer from severe infectious diseases, leading to their death or disability. Our purpose was developing of simple, inexpensive, high throughput technique based on the quantitative determination of TREC and KREC molecules by real-time PCR, and its validation in a group of children with a verified diagnosis of SCID and X-linked agammaglobulinemia.In this study, we developed and validated multiplex real-time PCR for the TREC’s and KREC’s quantitative analysis. We have shown that linear range of Ct changes depending on the concentrations of targets with a correlation coefficient R2 not worse than 0.98 was observed at concentrations from 109 to 5 × 104 copies per ml. The lowest amount of targets reliably detected in a reaction volume was 10 TREC’s copies, 5 KREC ‘s copies and 5 copies of internal control (IL17RA. We determined the age-depended reference values of TRECs and KRECs in whole blood in 29 boys and 27 girls with normal immunological parameters. The normal cut-offs for TRECs and KRECs were defined in dry blood spots depending on the method of extraction.The proposed method showed 100% diagnostic sensitivity and specificity in the studied group. The method can be proposed as a screening tool for the diagnosis of SCID and X-linked agammaglobulinemia both in whole blood and in the dry blood spots. The further investigation is required with larger number of samples. 

  9. Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids

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    Gasthuys Frank

    2006-04-01

    Full Text Available Abstract Background Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses. Results In the present study the gene transcription levels of 6 commonly used reference genes (ACTB, B2M, HPRT1, UBB, TUBA1 and RPL32 were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, TUBA1, ACTB and UBB were found to be most stable in normal skin and B2M, ACTB and UBB in equine sarcoids. Conclusion Based on these results, TUBA1, ACTB and UBB, respectively B2M, ACTB and UBB can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of UBB, ACTB and B2M can be recommended as a reliable and accurate normalisation factor.

  10. Nanodroplet real-time PCR system with laser assisted heating.

    Science.gov (United States)

    Kim, Hanyoup; Dixit, Sanhita; Green, Christopher J; Faris, Gregory W

    2009-01-01

    We report the successful application of low-power (approximately 30 mW) laser radiation as an optical heating source for high-speed real-time polymerase chain reaction (PCR) amplification of DNA in nanoliter droplets dispersed in an oil phase. Light provides the heating, temperature measurement, and Taqman real-time readout in nanoliter droplets on a disposable plastic substrate. A selective heating scheme using an infrared laser appears ideal for driving PCR because it heats only the droplet, not the oil or plastic substrate, providing fast heating and completing the 40 cycles of PCR in 370 seconds. No microheaters or microfluidic circuitry were deposited on the substrate, and PCR was performed in one droplet without affecting neighboring droplets. The assay performance was quantitative and its amplification efficiency was comparable to that of a commercial instrument.

  11. Identification and validation of reference genes for accurate normalization of real-time quantitative PCR data in kiwifruit.

    Science.gov (United States)

    Ferradás, Yolanda; Rey, Laura; Martínez, Óscar; Rey, Manuel; González, Ma Victoria

    2016-05-01

    Identification and validation of reference genes are required for the normalization of qPCR data. We studied the expression stability produced by eight primer pairs amplifying four common genes used as references for normalization. Samples representing different tissues, organs and developmental stages in kiwifruit (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev.) were used. A total of 117 kiwifruit samples were divided into five sample sets (mature leaves, axillary buds, stigmatic arms, fruit flesh and seeds). All samples were also analysed as a single set. The expression stability of the candidate primer pairs was tested using three algorithms (geNorm, NormFinder and BestKeeper). The minimum number of reference genes necessary for normalization was also determined. A unique primer pair was selected for amplifying the 18S rRNA gene. The primer pair selected for amplifying the ACTIN gene was different depending on the sample set. 18S 2 and ACT 2 were the candidate primer pairs selected for normalization in the three sample sets (mature leaves, fruit flesh and stigmatic arms). 18S 2 and ACT 3 were the primer pairs selected for normalization in axillary buds. No primer pair could be selected for use as the reference for the seed sample set. The analysis of all samples in a single set did not produce the selection of any stably expressing primer pair. Considering data previously reported in the literature, we validated the selected primer pairs amplifying the FLOWERING LOCUS T gene for use in the normalization of gene expression in kiwifruit.

  12. Quantitative monitoring of HCMV DNAlactia in human milk by real time PCR assay: Implementation of internal control contributes to standardization and quality control.

    Science.gov (United States)

    Hartleif, Steffen; Göhring, Katharina; Goelz, Rangmar; Jahn, Gerhard; Hamprecht, Klaus

    2016-11-01

    For cytomegalovirus screening of breastfeeding mothers of preterm infants under risk, we present a rapid, quantitative real-time PCR protocol using the hybridization format of the viral gB target region. For quantification, we used an external gB fragment cloned into a vector system. For standardization, we created an internal control-plasmid by site-directed mutagenesis with an exchange of 9 nucleotides. Spiked with internal control, patient wildtype amplicons could be discriminated from internal controls by hybridization probes using two-channel fluorescence detection. Potential bias of formerly reported false nucleotide sequence data of gB-hybridization probes was excluded. Using this approach, we could demonstrate excellent analytical performance and high reproducibility of HCMV detection during lactation. This assay shows very good correlation with a commercial quantitative HCMV DNA PCR and may help to identify rapidly HCMV shedding mothers of very low birth weight preterm infants to prevent HCMV transmission. On the other hand, negative DNA amplification results allow feeding of milk samples of seropositive mothers to their preterm infants under risk (<30 weeks of gestational age, <1000g birth weight) during the onset and late stage of HCMV shedding during lactation. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Stable Internal Reference Genes for Normalizing Real-Time Quantitative PCR in Baphicacanthus cusia under Hormonal Stimuli and UV Irradiation, and in Different Plant Organs

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    Yuxiang Huang

    2017-05-01

    Full Text Available Baphicacanthus cusia (Nees Bremek, the plant source for many kinds of drugs in traditional Chinese medicine, is widely distributed in South China, especially in Fujian. Recent studies about B. cusia mainly focus on its chemical composition and pharmacological effects, but further analysis of the plant's gene functions and expression is required to better understand the synthesis of its effective compounds. Real-time quantitative polymerase chain reaction (RT-qPCR is a powerful method for gene expression analysis. It is necessary to select a suitable reference gene for expression normalization to ensure the accuracy of RT-qPCR results. Ten candidate reference genes were selected from the transcriptome datasets of B. cusia in this study, and the expression stability was assessed across 60 samples representing different tissues and organs under various conditions, including ultraviolet (UV irradiation, hormonal stimuli (jasmonic acid methyl ester and abscisic acid, and in different plant organs. By employing different algorithms, such as geNorm, NormFinder, and BestKeeper, which are complementary approaches based on different statistical procedures, 18S rRNA was found to be the most stable gene under UV irradiation and hormonal stimuli, whereas ubiquitin-conjugating enzyme E2 was the best suitable gene for different plant organs. This novel study aimed to screen for suitable reference genes and corresponding primer pairs specifically designed for gene expression studies in B. cusia, in particular for RT-qPCR analyses.

  14. Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias.

    Science.gov (United States)

    Malik, Afshan N; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-08-19

    Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a "dilution bias" when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  15. Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.

    Science.gov (United States)

    Marcial-Quino, Jaime; Fierro, Francisco; De la Mora-De la Mora, Ignacio; Enríquez-Flores, Sergio; Gómez-Manzo, Saúl; Vanoye-Carlo, America; Garcia-Torres, Itzhel; Sierra-Palacios, Edgar; Reyes-Vivas, Horacio

    2016-04-25

    The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia.

  16. Enumeration of salmonellae in table eggs, pasteurized egg products, and egg-containing dishes by using quantitative real-time PCR.

    Science.gov (United States)

    Jakociune, Dziuginta; Pasquali, Frédérique; da Silva, Cristiana Soares; Löfström, Charlotta; Hoorfar, Jeffrey; Klein, Günter; Manfreda, Gerardo; Olsen, John Elmerdahl

    2014-03-01

    Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (Cq) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products.

  17. Comparative analysis of quantitative reverse transcription real-time PCR and commercial enzyme imunoassays for detection of enterotoxigenic Bacillus thuringiensis isolates.

    Science.gov (United States)

    Kaminska, Paulina S; Yernazarova, Aliya; Murawska, Emilia; Swiecicki, Jakub; Fiedoruk, Krzysztof; Bideshi, Dennis K; Swiecicka, Izabela

    2014-08-01

    Entomopathogenic Bacillus thuringiensis is closely related to Bacillus cereus, a human pathogen known to cause emesis and diarrhea. Standard detection methods do not distinguish these bacilli. Hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) genes that encode, respectively, HBL and NHE enterotoxins, are known to be harbored in both bacterial species, suggesting that differentiation of these bacilli is clinically and epidemiologically relevant. In this study the reliability of quantitative reverse transcription real-time PCR (qRT-PCR) and enzyme immunoassays (EIAs) in detecting hbl and nhe transcripts and corresponding toxins in environmental B. thuringiensis isolates was assessed. At least one enterotoxin gene was present in each isolate, and nhe or hbl genes were found in 85% and 55% of the strains, respectively. Based on statistical analyses, both BCET-RPLA and Duopath detected HBL at similar levels, and TECRA and Duopath can be used interchangeably for the detection of NHE, although TECRA has significantly lower sensitivity than Duopath. Thus, as potential enterotoxic B. thuringiensis strains occur in the natural environment, and EIA results may not correspond with the presence of enterotoxin genes and their expression, we suggest that reliable interpretation will be significantly enhanced by including qRT-PCR to support inferences based on EIAs.

  18. Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR.

    Science.gov (United States)

    Decorte, Inge; Van der Stede, Yves; Nauwynck, Hans; De Regge, Nick; Cay, Ann Brigitte

    2013-08-01

    This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser.

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

    Science.gov (United States)

    Gruselle, Olivier; Coche, Thierry; Louahed, Jamila

    2015-07-01

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

  20. Detection of anatid herpesvirus 1 gC gene by TaqMan™ fluorescent quantitative real-time PCR with specific primers and probe

    Directory of Open Access Journals (Sweden)

    Jia Renyong

    2010-02-01

    Full Text Available Abstract Background Anatid herpesvirus 1 (AHV-1 is known for the difficulty of monitoring and controlling, because it has a long period of asymptomatic carrier state in waterfowls. Furthermore, as a significant essential agent for viral attachment, release, stability and virulence, gC (UL44 gene and its protein product (glycoprotein C may play a key role in the epidemiological screening. The objectives of this study were to rapidly, sensitively, quantitatively detect gC gene of AHV-1 and provide the underlying basis for further investigating pcDNA3.1-gC DNA vaccine in infected ducks by TaqMan™ fluorescent quantitative real-time PCR assay (FQ-PCR with pcDNA3.1-gC plasmid. Results The repeatable and reproducible quantitative assay was established by the standard curve with a wide dynamic range (eight logarithmic units of concentration and very good correlation values (1.000. This protocol was able to detect as little as 1.0 × 101 DNA copies per reaction and it was highly specific to AHV-1. The TaqMan™ FQ-PCR assay successfully detected the gC gene in tissue samples from pcDNA3.1-gC and AHV-1 attenuated vaccine (AHV-1 Cha strain inoculated ducks respectively. Conclusions The assay offers an attractive method for the detection of AHV-1, the investigation of distribution pattern of AHV-1 in vivo and molecular epidemiological screening. Meanwhile, this method could expedite related AHV-1 and gC DNA vaccine research.

  1. The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Catríona M. Dowling

    2016-03-01

    Full Text Available Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR remains the most sensitive technique for nucleic acid quantification. Its popularity is reflected in the remarkable number of publications reporting RT-qPCR data. Careful normalisation within RT-qPCR studies is imperative to ensure accurate quantification of mRNA levels. This is commonly achieved through the use of reference genes as an internal control to normalise the mRNA levels between different samples. The selection of appropriate reference genes can be a challenge as transcript levels vary with physiology, pathology and development, making the information within the transcriptome flexible and variable. In this study, we examined the variation in expression of a panel of nine candidate reference genes in HCT116 and HT29 2-dimensional and 3-dimensional cultures, as well as in normal and cancerous colon tissue. Using normfinder we identified the top three most stable genes for all conditions. Further to this we compared the change in expression of a selection of PKC coding genes when the data was normalised to one reference gene and three reference genes. Here we demonstrated that there is a variation in the fold changes obtained dependent on the number of reference genes used. As well as this, we highlight important considerations namely; assay efficiency tests, inhibition tests and RNA assessment which should also be implemented into all RT-qPCR studies. All this data combined demonstrates the need for careful experimental design in RT-qPCR studies to help eliminate false interpretation and reporting of results.

  2. The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Catríona M Dowling

    2016-01-01

    Full Text Available Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR remains the most sensitive technique for nucleic acid quantification. Its popularity is reflected in the remarkable number of publications reporting RT-qPCR data. Careful normalisation within RT-qPCR studies is imperative to ensure accurate quantification of mRNA levels. This is commonly achieved through the use of reference genes as an internal control to normalise the mRNA levels between different samples. The selection of appropriate reference genes can be a challenge as transcript levels vary with physiology, pathology and development, making the information within the transcriptome flexible and variable. In this study, we examined the variation in expression of a panel of nine candidate reference genes in HCT116 and HT29 2-dimensional and 3-dimensional cultures, as well as in normal and cancerous colon tissue. Using normfinder we identified the top three most stable genes for all conditions. Further to this we compared the change in expression of a selection of PKC coding genes when the data was normalised to one reference gene and three reference genes. Here we demonstrated that there is a variation in the fold changes obtained dependent on the number of reference genes used. As well as this, we highlight important considerations namely; assay efficiency tests, inhibition tests and RNA assessment which should also be implemented into all RT-qPCR studies. All this data combined demonstrates the need for careful experimental design in RT-qPCR studies to help eliminate false interpretation and reporting of results.

  3. Detection of live Salmonella sp. cells in produce by a TaqMan-based quantitative reverse transcriptase real-time PCR targeting invA mRNA.

    Science.gov (United States)

    González-Escalona, Narjol; Hammack, Thomas S; Russell, Mindi; Jacobson, Andrew P; De Jesús, Antonio J; Brown, Eric W; Lampel, Keith A

    2009-06-01

    Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 10(3) CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 10(5) and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDA's Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/ approximately ebam/bam-5.html, 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers.

  4. Selection of reference genes for quantitative real-time PCR normalization in Panax ginseng at different stages of growth and in different organs.

    Science.gov (United States)

    Liu, Jing; Wang, Qun; Sun, Minying; Zhu, Linlin; Yang, Michael; Zhao, Yu

    2014-01-01

    Quantitative real-time reverse transcription PCR (qRT-PCR) has become a widely used method for gene expression analysis; however, its data interpretation largely depends on the stability of reference genes. The transcriptomics of Panax ginseng, one of the most popular and traditional ingredients used in Chinese medicines, is increasingly being studied. Furthermore, it is vital to establish a series of reliable reference genes when qRT-PCR is used to assess the gene expression profile of ginseng. In this study, we screened out candidate reference genes for ginseng using gene expression data generated by a high-throughput sequencing platform. Based on the statistical tests, 20 reference genes (10 traditional housekeeping genes and 10 novel genes) were selected. These genes were tested for the normalization of expression levels in five growth stages and three distinct plant organs of ginseng by qPCR. These genes were subsequently ranked and compared according to the stability of their expressions using geNorm, NormFinder, and BestKeeper computational programs. Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples. GAPDH/30S RPS20, CYP/60S RPL13 and CYP/QCR were the optimum pair of reference genes in the roots, stems, and leaves. CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages. Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng.

  5. Evaluation of Real-Time Quantitative Polymerase Chain Reaction (qPCR) to Determine Escherichia coli Concentrations at Two Lake Erie Beaches

    Science.gov (United States)

    Kephart, Christopher M.; Bushon, Rebecca N.

    2009-01-01

    During the recreational seasons of 2006 and 2007, the quantitative polymerase chain reaction (qPCR) method was used to determine Escherichia coli (E. coli) concentrations in samples from two Lake Erie beaches. Results from the qPCR method were compared to those obtained by traditional culturing on modified mTEC agar. Regression analysis showed strong, statistically significant correlations between results from the two methods for both years. Correlation coefficients at Edgewater and Villa Angela Beaches were 0.626 and 0.789 for 2006 and 0.667 and 0.829 for 2007, respectively. Linear regression analyses were done to determine how well E. coli concentrations could have been predicted from qPCR results. These hypothetical predictions were compared to the current practice of determining recreational water quality from E. coli concentrations determined for samples collected on the previous day. The qPCR method resulted in a greater percentage of correct predictions of water-quality exceedances than the current method for both beaches and both years. However, because regression equations differed somewhat between both sites and both years, the study did not result in any single relation reliable enough to use for actual real-time prediction of water-quality exceedances for either beach; therefore, a posterior analysis of data was done. Additional years of data may be needed to develop such a relation. Results from this study support the continued development and testing of a qPCR method for providing rapid and accurate estimates of E. coli concentrations for monitoring recreational water quality.

  6. Estimating the Sensitivity and Specificity of Real-Time Quantitative PCR of Fecal Samples for Diagnosis of Rhodococcus equi Pneumonia in Foals.

    Science.gov (United States)

    Shaw, S D; Cohen, N D; Chaffin, M K; Blodgett, G P; Syndergaard, M; Hurych, D

    2015-01-01

    Real-time, quantitative PCR (qPCR) methods for detecting Rhodococcus equi in feces have been developed as a noninvasive, rapid diagnostic test for R. equi pneumonia, but have not been evaluated in a large population of foals. The objective of this study was to evaluate the clinical utility of fecal PCR as a diagnostic test for R. equi pneumonia in foals using receiver operating characteristic (ROC) methods. 186 foals born in 2011 at an R. equi-endemic ranch in Texas. Fecal samples were collected at the time of onset of clinical signs for pneumonic foals (n = 31). Foals with pneumonia were matched by age and birth date to healthy (n = 31) and subclinical (n = 124) control foals; fecal samples were collected from these controls. DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR. Concentration of R. equi in feces differed significantly (P equi pneumonia was 89% (95% CI, 83-99), with a sensitivity of 94% and specificity of 72%. qPCR of feces can be useful as an alternative to tracheobronchial aspiration for the diagnosis of R. equi in foals with clinical signs of pneumonia. Caution should be used in extrapolating results of this study to other populations because fecal concentration of R. equi might vary by geographic location or management practices. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  7. A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Stewart Don

    2008-05-01

    Full Text Available Abstract Background Based upon defining a common reference point, current real-time quantitative PCR technologies compare relative differences in amplification profile position. As such, absolute quantification requires construction of target-specific standard curves that are highly resource intensive and prone to introducing quantitative errors. Sigmoidal modeling using nonlinear regression has previously demonstrated that absolute quantification can be accomplished without standard curves; however, quantitative errors caused by distortions within the plateau phase have impeded effective implementation of this alternative approach. Results Recognition that amplification rate is linearly correlated to amplicon quantity led to the derivation of two sigmoid functions that allow target quantification via linear regression analysis. In addition to circumventing quantitative errors produced by plateau distortions, this approach allows the amplification efficiency within individual amplification reactions to be determined. Absolute quantification is accomplished by first converting individual fluorescence readings into target quantity expressed in fluorescence units, followed by conversion into the number of target molecules via optical calibration. Founded upon expressing reaction fluorescence in relation to amplicon DNA mass, a seminal element of this study was to implement optical calibration using lambda gDNA as a universal quantitative standard. Not only does this eliminate the need to prepare target-specific quantitative standards, it relegates establishment of quantitative scale to a single, highly defined entity. The quantitative competency of this approach was assessed by exploiting "limiting dilution assay" for absolute quantification, which provided an independent gold standard from which to verify quantitative accuracy. This yielded substantive corroborating evidence that absolute accuracies of ± 25% can be routinely achieved. Comparison

  8. Quantitative real-time imaging of glutathione

    Science.gov (United States)

    Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time ...

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

    Directory of Open Access Journals (Sweden)

    Chun-Sun Gu

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  11. Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays.

    Science.gov (United States)

    Salehi, Elham; Hedayati, Mohammad T; Zoll, Jan; Rafati, Haleh; Ghasemi, Maryam; Doroudinia, Atosa; Abastabar, Mahdi; Tolooe, Ali; Snelders, Eveline; van der Lee, Henrich A; Rijs, Antonius J M M; Verweij, Paul E; Seyedmousavi, Seyedmojtaba; Melchers, Willem J G

    2016-11-01

    In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4- to 5-μm FFPE tissue sections from each specimen were digested with proteinase K, followed by automated nucleic acid extraction. Multiple real-time quantitative PCR (qPCR) assays targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, using fluorescently labeled primers, was performed to identify clinically important genera and species of Aspergillus, Fusarium, Scedosporium, and the Mucormycetes The molecular identification was correlated with results from histological examination. One of the main findings of our study was the high sensitivity of the automated DNA extraction method, which was estimated to be 94%. The qPCR procedure that was evaluated identified a range of fungal genera/species, including Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Fusarium oxysporum, Fusarium solani, Scedosporium apiospermum, Rhizopus oryzae, Rhizopus microsporus, Mucor spp., and Syncephalastrum Fusarium oxysporum and F. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis. Aspergillus flavus, S. apiospermum, and Syncephalastrum were detected from histopathological mucormycosis samples. In addition, examination of four samples from patients suspected of having concomitant aspergillosis and mucormycosis infections resulted in the identification of two A. flavus isolates, one Mucor isolate, and only one sample having both R. oryzae and A. flavus Our results indicate that histopathological features of molds may be easily confused in tissue sections. The qPCR assay used in this study is a reliable tool for the rapid and accurate identification of fungal pathogens to the genus and species levels directly from FFPE tissues. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    Directory of Open Access Journals (Sweden)

    Jing Cai

    Full Text Available Quantitative real-time PCR (qPCR is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD, an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA and nonparametric (Kruskal-Wallis tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  13. Selection and Validation of Reference Genes for Real-Time Quantitative PCR in Hyperaccumulating Ecotype of Sedum alfredii under Different Heavy Metals Stresses

    Science.gov (United States)

    Liu, Mingying; Qiao, Guirong; Jiang, Jing; Zhuo, Renying

    2013-01-01

    Real-time Quantitative PCR (RT-qPCR) has become an effective method for accurate analysis of gene expression in several biological systems as well as under different experimental conditions. Although with high sensitivity, specificity and broad dynamic range, this method requires suitable reference genes for transcript normalization in order to guarantee reproducible and meaningful results. In the present study, we evaluated five traditional housekeeping genes and five novel reference genes in Hyperaccumulating ecotype of Sedum alfredii, a well known hyperaccumulator for heavy metals phytoremediation, under Cd, Pb, Zn and Cu stresses of seven different durations. The expression stability of these ten candidates were determined with three programs - geNorm, NormFinder and BestKeeper. The results showed that all the selected reference genes except for SAND could be used for RT-qPCR normalization. Among them UBC9 and TUB were ranked as the most stable candidates across all samples by three programs together. For the least stable reference genes, however, BestKeeper produced different results compared with geNorm and NormFinder. Meanwhile, the expression profiles of PCS under Cd, Pb, Zn and Cu stresses were assessed using UBC9 and TUB respectively, and similar trends were obtained from the results of the two groups. The distinct expression patterns of PCS indicated that various strategies could be taken by plants in adaption to different heavy metals stresses. This study will provide appropriate reference genes for further gene expression quantification using RT-qPCR in Hyperaccumulator S. alfredii. PMID:24340067

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

    Science.gov (United States)

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

    2013-04-15

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

  15. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    Science.gov (United States)

    Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  16. Quantitative PCR - new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situhybridization

    DEFF Research Database (Denmark)

    Schlemmer, B.O.; Sørensen, B. S.; Overgaard, J.

    2004-01-01

    Previously, polymerase chain reaction (PCR) technology has been hampered by its inability to generate quantitative results, a drawback inherent to the high degree of amplification taking place in the reaction. Recently, PCR techniques have been described with the potential of quantifying the amount...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression......, and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real-time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained...

  17. 鹦鹉热衣原体real-time quantitative PCR检测方法的研究%Study on the Detection Method of Chlamydophila Psittaci by Using Real-time Quantitative PCR

    Institute of Scientific and Technical Information of China (English)

    吴东海; 李应国; 杨迎伍; 邓伟; 王昱; 李正国

    2008-01-01

    [目的]为鹦鹉热衣原体的快速、准确检测奠定基础.[方法]根据鹦鹉热衣原体主要外膜蛋白(MOMP)基因序列设计一对特异性引物,以SYBR GreenI为荧光染料,建立了鹦鹉热衣原体Real-time quantitative PCR检测方法.根据检测结果,计算样品的批内和批间变异系数(CV).[结果]以提取的鹦鹉热表原体DNA为模板,用引物Cps-1和Cps-2进行PCR扩增,得到长100 bp的片段.扩增产物回收纯化后,连接到pGEM-T Easy载体上并转化到大肠杆菌DH5α,菌落PCR检测筛选阳性克隆,培养后提取质粒DNA.当模板浓度范围为1.78×102~1.78×108拷贝/μl时,标准曲线相关系数达0.998;批内和批间变异系数(CV%)分别为1.30%~4.59%和5.72%~9.87%.[结论]为鹦鹉热衣原体的感染、流行调查等提供了重要的技术参考.

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

    Science.gov (United States)

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

    2013-06-01

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

  19. Evaluation and subsequent optimizations of the quantitative AmpliSens Florocenosis/Bacterial vaginosis-FRT multiplex real-time PCR assay for diagnosis of bacterial vaginosis.

    Science.gov (United States)

    Rumyantseva, Tatiana; Shipitsyna, Elena; Guschin, Alexander; Unemo, Magnus

    2016-12-01

    Traditional microscopy-based methods for diagnosis of bacterial vaginosis (BV) are underutilized in many settings, and molecular techniques may provide opportunities for rapid, objective, and accurate BV diagnosis. This study evaluated the quantitative AmpliSens Florocenosis/Bacterial vaginosis-FRT multiplex real-time PCR (Florocenosis-BV) assay. Vaginal samples from a previous study including unselected female subjects (n = 163) and using Amsel criteria and 454 pyrosequencing for BV diagnosis were examined with the Florocenosis-BV test and additionally tested for the presence and quantity of Gardnerella vaginalis clades 3 and 4. The Florocenosis-BV assay demonstrated 100% and 98% sensitivity compared with the Amsel criteria and 454 pyrosequencing, respectively, with 91% specificity. The modified Florocenosis-BV assay (detecting also G. vaginalis clades 3 and 4) resulted in 100% sensitivity vs the Amsel criteria and 454 pyrosequencing with specificity of 86% and 88%, respectively. Further optimizations of thresholds for the quantitative parameters used in the kit resulted in 99-100% accuracy vs Amsel criteria and 454 pyrosequencing for selected parameters. The Florocenosis-BV assay is an objective, accurate, sensitive, and specific method for BV diagnosis; however, the performance of the test can be further improved with some minor optimizations. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  20. Quantitation of infectious myonecrosis virus in different tissues of naturally infected Pacific white shrimp, Litopenaeus vannamei, using real-time PCR with SYBR Green chemistry.

    Science.gov (United States)

    da Silva, Suzianny Maria Bezerra Cabral; Pinheiro, Ana Cristina de Aguiar Saldanha; Coimbra, Maria Raquel Moura

    2011-11-01

    The Pacific white shrimp, Litopenaeus vannamei, is the most important shrimp species in volume in world aquaculture. However, in recent decades, outbreaks of diseases, especially viral diseases, have led to significant economic losses, threatening the sustainability of shrimp farming worldwide. In 2004, Brazilian shrimp farming was seriously affected by a new disease caused by the Infectious myonecrosis virus (IMNV). Thus, disease control based on rapid and sensitive pathogen detection methods has become a priority. In this study, a specific quantitation method for IMNV was developed using real-time PCR with SYBR Green chemistry and viral load of the principal target tissues of chronically infected animals was quantified. The quantitative analysis revealed that mean viral load ranged from 5.08×10(8) to 1.33×10(6)copies/μg of total RNA in the hemolymph, 5.096×10(5) to 1.26×10(3)copies/μg in the pleopods, 6.85×10(8) to 3.09×10(4)copies/μg in muscle and 8.15×10(6) to 3.90×10(3)copies/μg in gills. Different viral loads of IMNV were found with greater values in the hemolymph and muscle, followed by the pleopods and gills.

  1. 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...

  2. Diagnosing Smith-Magenis syndrome and duplication 17p11.2 syndrome by RAI1 gene copy number variation using quantitative real-time PCR.

    Science.gov (United States)

    Truong, Hoa T; Solaymani-Kohal, Sara; Baker, Kevin R; Girirajan, Santhosh; Williams, Stephen R; Vlangos, Christopher N; Smith, Ann C M; Bunyan, David J; Roffey, Paul E; Blanchard, Christopher L; Elsea, Sarah H

    2008-03-01

    Smith-Magenis syndrome (SMS) and duplication 17p11.2 (dup17p11.2) syndrome are multiple congenital anomalies/mental retardation disorders resulting from either a deletion or duplication of the 17p11.2 region, respectively. The retinoic acid induced 1 (RAI1) gene is the causative gene for SMS and is included in the 17p11.2 region of dup17p11.2 syndrome. Currently SMS and dup17p11.2 syndrome are diagnosed using a combination of clinically recognized phenotypes and molecular cytogenetic analyses such as fluorescent in situ hybridization (FISH). However, these methods have proven to be highly expensive, time consuming, and dependent upon the low resolving capabilities of the assay. To address the need for improved diagnostic methods for SMS and dup17p11.2 syndrome, we designed a quantitative real-time PCR (Q-PCR) assay that measures RAI1 copy number using the comparative C(t) method, DeltaDeltaC(t). We tested our assay with samples blinded to their previous SMS or dup17p11.2 syndrome status. In all cases, we were able to determine RAI1 copy number status and render a correct diagnosis accordingly. We validated these results by both FISH and multiplex ligation-dependent probe amplification (MLPA). We conclude that Q-PCR is an accurate, reproducible, low-cost, and reliable assay that can be employed for routine use in SMS and dup17p11.2 diagnosis.

  3. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    Energy Technology Data Exchange (ETDEWEB)

    Malik, Afshan N., E-mail: afshan.malik@kcl.ac.uk [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom); Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom)

    2011-08-19

    Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as {beta}-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  4. Quantitative Real-Time PCR Analysis of YKL-40 and Its Comparison with Mammalian Chitinase mRNAs in Normal Human Tissues Using a Single Standard DNA

    Directory of Open Access Journals (Sweden)

    Misa Ohno

    2015-04-01

    Full Text Available YKL-40 (YKL for the first three N-terminal residues of a 40 kDa protein belongs to a group of human chitinase-like proteins (CLPs, which are similar to chitinases but lack chitinolytic activity. YKL-40 mRNA and its protein levels have been reported elevated in multiple disorders including asthma, cystic fibrosis, rheumatoid arthritis and malignant tumors. Here, we quantified the YKL-40 mRNA levels and compared them with chitinases and housekeeping genes in normal human tissues. To establish the quantitative real-time PCR (qPCR system for evaluation of relative YKL-40 mRNA levels, we constructed a human standard DNA molecule by ligating cDNAs of YKL-40, two mammalian chitinases and two housekeeping genes in a one-to-one ratio. We generated cDNAs from various normal human tissues and analyzed the YKL-40 mRNA expression levels using a qPCR system with the standard DNA. We found that YKL-40 mRNA is present widely in human tissues while its expression patterns exhibit clear tissue specificity. Highest YKL-40 mRNA levels were detected in the liver, followed by kidney, trachea and lung. The levels of YKL-40 mRNA in the kidney and liver were more than 100-times higher than those of chitotriosidase mRNA. Our study provides for the first time a comprehensive analysis of the relative expression levels of YKL-40 mRNA versus mammalian chitinases in normal human tissues.

  5. Entomopathogenic nematodes, phoretic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs in Florida citrus groves.

    Science.gov (United States)

    Campos-Herrera, Raquel; El-Borai, Fahiem E; Stuart, Robin J; Graham, James H; Duncan, Larry W

    2011-09-01

    Quantitative real-time PCR (qPCR) is a powerful tool to detect and quantify species of cryptic organisms such as bacteria, fungi and nematodes from soil samples. As such, qPCR offers new opportunities to study the ecology of soil habitats by providing a single method to characterize communities of diverse organisms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Paenibacillus nematophilus and Paenibacillus sp.) phoretically associated with entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematodae. We also extend the repertoire of species specific primers and TaqMan® probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commercially for biological control. Primers and probes were designed from the ITS rDNA region for the EPNs and the 16S rDNA region for the bacteria. Standard curves were established using DNA from pure cultures of EPNs and plasmid DNA from the bacteria. The use of TaqMan probes in qPCR resolved the non-specificity of EPN and some bacterial primer amplifications whereas those for Paenibacillus sp. also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two species that are not phoretically associated with nematodes. The primer-probe sets for EPNs were able to accurately detect three infective juvenile EPNs added to nematodes recovered from soil samples. The molecular set for Paenibacillus sp. detected the bacterium attached to Steinernema diaprepesi suspended in water or added to nematodes recovered from soil samples but its detection decreased markedly in the soil samples, even when a nested PCR protocol was employed. Using qPCR we detected S. scapterisci at low levels in a citrus grove, which suggested natural long-distance spread of this exotic species, which is applied to pastures and golf courses to manage mole crickets (Scapteriscus spp

  6. Development of a quantitative Real-Time PCR for micrometastasis detection using CEA in peripheral blood and bone marrow specimens of gastric cancer patients

    Directory of Open Access Journals (Sweden)

    Dardaei Alghalandis L

    2009-11-01

    transcription and subjected to Quantitative Real-Time PCR (QRT-PCR."n"nResults: We developed a highly sensitive and specific quantitative PCR for CEA and GAPDH using Real-Time PCR based on TaqMan technology. CEA mRNA was detected in 23% of PB and 20% of BM specimens. There was no CEA mRNA detecting in control group."n"nConclusions: The QRT-PCR for CEA can be a useful technique for detection of micrometastases in the PB and BM specimens of gastric cancer patients."n

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  8. Optimization and validation of DNA extraction and real-time PCR assay for the quantitative measurement of residual host cell DNA in biopharmaceutical products.

    Science.gov (United States)

    Hu, B; Sellers, J; Kupec, J; Ngo, W; Fenton, S; Yang, T-Y; Grebanier, A

    2014-01-01

    Host cell DNA contamination occurs during the production of biopharmaceuticals and must be controlled and monitored for the purity and safety of the drug products. A sodium iodide-based DNA extraction and a subsequent real time PCR assay were developed and validated for the quantitative measurement of residual host cell DNA impurity in monoclonal antibody therapeutic products. A sodium iodide-based commercial kit was optimized for the removal of interfering matrices. Several incubation steps from the kit protocol were combined and a neutralization buffer was introduced to protein digestion step to eliminate any precipitation from the detergent. The elimination of the two washing steps significantly reduced assay variability from loss of DNA pellets. The optimized DNA extraction procedure can recover DNA close to 100% for DNA concentrations from 10 to 100,000pg/mL. Of the published sequences of repetitive interspersed nuclear elements, we identified a nucleotide mismatch from the published CHO probe. Correction of this nucleotide increased DNA amplification by a thousand fold. The optimized assay was further validated for the quantitation of residual CHO DNA according to ICH guidelines with preset assay acceptance criteria. The method met all assay acceptance criteria and was found linear, accurate and precise for the quantitation of residual CHO in the linear range of 10-100,000pg DNA/mL. LOQ was measured at 10pg DNA/mL and LOD at 1pg DNA/mL. No matrix interference to our validated assay was detected from bioreactor harvest, Protein A eluate or eluate from ion exchange columns.

  9. Validation and application of a quantitative real-time PCR assay to detect common wheat adulteration of durum wheat for pasta production.

    Science.gov (United States)

    Carloni, Elisa; Amagliani, Giulia; Omiccioli, Enrica; Ceppetelli, Veronica; Del Mastro, Michele; Rotundo, Luca; Brandi, Giorgio; Magnani, Mauro

    2017-06-01

    Pasta is the Italian product par excellence and it is now popular worldwide. Pasta of a superior quality is made with pure durum wheat. In Italy, addition of Triticum aestivum (common wheat) during manufacturing is not allowed and, without adequate labeling, its presence is considered an adulteration. PCR-related techniques can be employed for the detection of common wheat contaminations. In this work, we demonstrated that a previously published method for the detection of T. aestivum, based on the gliadin gene, is inadequate. Moreover, a new molecular method, based on DNA extraction from semolina and real-time PCR determination of T. aestivum in Triticum spp., was validated. This multiplex real-time PCR, based on the dual-labeled probe strategy, guarantees target detection specificity and sensitivity in a short period of time. Moreover, the molecular analysis of common wheat contamination in commercial wheat and flours is described for the first time.

  10. Evaluation of imatinib mesylate(Gleevec) on KAI1/CD82 gene expression in breast cancer MCF-7 cells using quantitative real-time PCR

    Institute of Scientific and Technical Information of China (English)

    Seyed Ataollah Sadat Shandiz; Marjan Khosravani; Sepideh Mohammadi; Hassan Noorbazargan; Amir Mirzaie; Davoud Nouri Inanlou; Mojgan Dalirsaber Jalali; Hamidreza Jouzaghkar; Fahimeh Baghbani-Arani; Behta Keshavarz-Pakseresht

    2016-01-01

    Objective: To evaluate the effect of imatinib mesylate on cell viability, anti cancer effect through modulation of KAI1/CD82 gene expression in breast cancer MCF-7 cell line.Methods: The effects of imatinib mesylate on cell viability in MCF-7 cell line were assessed using MTT assay and IC50 value was determined. GAPDH and KAI1/CD82 were selected as reference and target genes, respectively. Quantitative real time PCR technique was applied for investigation of KAI1/CD82 gene expression in human breast cancer MCF-7 cells. Subsequently, the quantity of KAI1 compared to GAPDH gene expressions were analyzed using the formula; 2-DDCt.Results: Imatinib was showed to have a dose-dependent inhibitory effect on the viability of MCF-7 cells. CD82/GAPDH gene expression ratios were 1.322 ± 0.030(P > 0.05),2.052 ± 0.200(P < 0.05), 2.151 ± 0.270(P < 0.05) for 10, 20 and 40 mmol/L of imatinib concentrations.Conclusions: Based on the present data, imatinib mesylate might modulate metastasis by up-regulating KAI1/CD82 gene expression in human breast MCF-7 cancer cell line.

  11. Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole (Cynoglossus semilaevis)

    Institute of Scientific and Technical Information of China (English)

    MA Qian; ZHUANG Zhimeng; FENG Wenrong; LIU Shufang; TANG Qisheng

    2015-01-01

    Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization duringCynoglossus semilaevis early developmental process, eight candidate reference genes (ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S) were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the matureC.semilaevis. EF1A was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages (from oosphere to 90 days old) in this study. However, when divided theCt value data of the above mentioned early developmental stages into two separate periods (embryo and post-hatching periods), TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.

  12. Differentially expressed genes of Tetrahymena thermophila in response to tributyltin (TBT) identified by suppression subtractive hybridization and real time quantitative PCR.

    Science.gov (United States)

    Feng, Lifang; Miao, Wei; Wu, Yuxuan

    2007-02-15

    Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T. thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system.

  13. Quantitative Real-Time Reverse Transcription-PCR Assay for the Expression of Tob mRNA in Human Colorectal Cancer

    Institute of Scientific and Technical Information of China (English)

    Dian-chao WU

    2010-01-01

    OBJECTIVE Tob is a member of Tob/BTG antiproliferative family. To date, Tob expression in human carcinoma using clinical specimens has not been studied in depth except for lung carcinoma and thyroid carcinoma. This study is the first to investigate the expression levels of Tob gene in human colorectal cancer tissues,and their corresponding para-cancerous tissues. The correlation of expression of the Tob gene with clinicopathological characteristics of colorectal cancer was also analyzed.METHODS Quantitative real time RT-PCR was used to detect the expression of Tob mRNA in 31 colorectal cancers.RESULTS Compared with normal tissues, up-regulation of Tob mRNA was observed in 31 colorectal cancer tissues (P = 0.020).The expression level of Tob at Dukes C + D phase was higher than Dukes A + B phase, and the difference was signifi cant (P < 0.05).However, in this study, it was found that the expression of Tob mRNA was not related with age, gender, and pathological type of colorectal cancer.CONCLUSION The up-regulation of Tob may be closely associated with tumorigenesis of colorectal carcinoma.

  14. Analysis of reference gene expression for real-time PCR based on relative quantitation and dual spike-in strategy in the silkworm Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    Ran Peng; Yuanfen Zhai; Hua Ding; Tianyuan Di; Ting Zhang; Bing Li; Weide Shen; Zhengguo Wei

    2012-01-01

    In general,for real-time quantitative polymerase chain reaction (qPCR),normalization strategies use a reference gene as a control and to avoid the introduction of experi-mental errors expression of this gene should not vary in response to changing conditions.However,the expression of many reference genes has been reported to vary consid-erably and,without appropriate normalization,the expression profile of a target gene can be misinterpreted.In this study,the expression levels of seven commonly used reference genes (ACT,GAPDH,28srRNA,RPL3,α-tubulin,UBC,and TBP) were detected at different development time points and in response to treatment with 20-hydroxyecdysone (20E) and with rntin.The expression stability was analyzed using geNorm and NormFinder software.Significant variations were found among normal tissues and between experimentally treated tissues.The dual spike-in strategy also revealed significant variations of the expression levels of the reference genes among normal tissues and between experimentally treated tissues.Glutathione-S-transferase sigma 1 (GSTs1),which has a high expression level in fat body and is related to the mechanism of resistance,was used as a target gene to validate the feasibility and difference of these two approaches.

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

    Institute of Scientific and Technical Information of China (English)

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

    2016-01-01

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

  16. Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR.

    Science.gov (United States)

    Du, Yishuai; Zhang, Linlin; Xu, Fei; Huang, Baoyu; Zhang, Guofan; Li, Li

    2013-03-01

    Hatchery-reared larvae of the Pacific oyster (Crassostrea gigas) often suffer from massive mortality induced by Ostreid herpesvirus 1 (OsHV-1) infection, indicating the importance of better understanding of oyster immune defense systems. The accuracy of measurements of gene expression levels based on quantitative real-time PCR assays relies on the use of housekeeping genes as internal controls; however, few studies have focused on the selection of such internal controls. In this study, we conducted a comprehensive investigation of internal control genes during oyster development in virus-infected and uninfected samples. Transcriptome data for 38 developmental stages were downloaded and the gene expression patterns were classified into 30 clusters. A total of 317 orthologs of classical housekeeping genes in the oyster genome were annotated. After combining the expression profiles and oyster housekeeping gene dataset, 14 candidate internal controls were selected for further investigation: Elongation factor-1α (EF-1α), 18S rRNA (18S), 28S rRNA (28S), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin (ACT), Ribosomal protein L7 (RL7), Ribosomal protein L27 (RL27), Ribosomal protein L36 (RL36), Ribosomal protein S18 (RS18), Heterogeneous nuclear ribonucleoprotein A2/B1 (RO21), Eukaryotic translation elongation factor 2 (EF2), Ubiquitin-conjugating enzyme E2D2 (UBCD1), S-phase kinase-associated protein 1 (SKP1) and Heterogeneous nuclear ribonucleoprotein Q (HNRPQ). RNA was extracted from oyster larvae infected with OsHV-1 (group A; GA), and OsHV-1 free larvae (group B; GB). The expression levels of the 14 candidate internal controls were studied in GA and GB larvae by real-time PCR. Their expression stabilities were further analyzed using the GeNorm program. RL7 and RS18 were the most stable genes in both OsHV-1 infected (GA) and uninfected (GB) larvae. These results suggest that RL7 and RS18 could be used as internal controls for studying gene expression in

  17. Real-Time PCR for Universal Phytoplasma Detection and Quantification

    DEFF Research Database (Denmark)

    Christensen, Nynne Meyn; Nyskjold, Henriette; Nicolaisen, Mogens

    2013-01-01

    Currently, the most efficient detection and precise quantification of phytoplasmas is by real-time PCR. Compared to nested PCR, this method is less sensitive to contamination and is less work intensive. Therefore, a universal real-time PCR method will be valuable in screening programs and in other...

  18. 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.

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

    Science.gov (United States)

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

    2013-09-15

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

  20. Highly sensitive detection of the group A Rotavirus using Apolipoprotein H-coated ELISA plates compared to quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Veas Francisco

    2011-02-01

    Full Text Available Abstract Background The principle of a capture ELISA is binding of specific capture antibodies (polyclonal or monoclonal to the surface of a suitable 96 well plate. These immobilized antibodies are capable of specifically binding a virus present in a clinical sample. Subsequently, the captured virus is detected using a specific detection antibody. The drawback of this method is that a capture ELISA can only function for a single virus captured by the primary antibody. Human Apolipoprotein H (ApoH or β2-glycoprotein 1 is able to poly-specifically bind viral pathogens. Replacing specific capture antibodies by ApoH should allow poly-specific capture of different viruses that subsequently could be revealed using specific detection antibodies. Thus, using a single capture ELISA format different viruses could be analysed depending on the detection antibody that is applied. In order to demonstrate that this is a valid approach we show detection of group A rotaviruses from stool samples as a proof of principle for a new method of capture ELISA that should also be applicable to other viruses. Results Stool samples of different circulating common human and potentially zoonotic group A rotavirus strains, which were pretested in commercial EIAs and genotyped by PCR, were tested in parallel in an ApoH-ELISA set-up and by quantitative real-time PCR (qPCR. Several control samples were included in the analysis. The ApoH-ELISA was suitable for the capture of rotavirus-particles and the detection down to 1,000 infectious units (TCID50/ml. Subsets of diagnostic samples of different G- and P-types were tested positive in the ApoH-ELISA in different dilutions. Compared to the qPCR results, the analysis showed high sensitivity, specificity and low cross-reactivity for the ApoH-ELISA, which was confirmed in receiver operating characteristics (ROC analysis. Conclusions In this study the development of a highly sensitive and specific capture ELISA was demonstrated by

  1. Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions.

    Science.gov (United States)

    Imai, Tsuyoshi; Ubi, Benjamin E; Saito, Takanori; Moriguchi, Takaya

    2014-01-01

    We have evaluated suitable reference genes for real time (RT)-quantitative PCR (qPCR) analysis in Japanese pear (Pyrus pyrifolia). We tested most frequently used genes in the literature such as β-Tubulin, Histone H3, Actin, Elongation factor-1α, Glyceraldehyde-3-phosphate dehydrogenase, together with newly added genes Annexin, SAND and TIP41. A total of 17 primer combinations for these eight genes were evaluated using cDNAs synthesized from 16 tissue samples from four groups, namely: flower bud, flower organ, fruit flesh and fruit skin. Gene expression stabilities were analyzed using geNorm and NormFinder software packages or by ΔCt method. geNorm analysis indicated three best performing genes as being sufficient for reliable normalization of RT-qPCR data. Suitable reference genes were different among sample groups, suggesting the importance of validation of gene expression stability of reference genes in the samples of interest. Ranking of stability was basically similar between geNorm and NormFinder, suggesting usefulness of these programs based on different algorithms. ΔCt method suggested somewhat different results in some groups such as flower organ or fruit skin; though the overall results were in good correlation with geNorm or NormFinder. Gene expression of two cold-inducible genes PpCBF2 and PpCBF4 were quantified using the three most and the three least stable reference genes suggested by geNorm. Although normalized quantities were different between them, the relative quantities within a group of samples were similar even when the least stable reference genes were used. Our data suggested that using the geometric mean value of three reference genes for normalization is quite a reliable approach to evaluating gene expression by RT-qPCR. We propose that the initial evaluation of gene expression stability by ΔCt method, and subsequent evaluation by geNorm or NormFinder for limited number of superior gene candidates will be a practical way of finding out

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

    Directory of Open Access Journals (Sweden)

    Kundu Jiban K

    2010-07-01

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

  3. Principle and Quality Control of Real-time Fluorescent Quantitative PCR%实时荧光定量 PCR 仪原理与技术关键点分析

    Institute of Scientific and Technical Information of China (English)

    郑沁春

    2012-01-01

      Expounds the basic constitution and measurement principle of Real-time fluorescent quantitative PCR.Describes briefly the quality control and the factors.%  本文主要阐述实时荧光定量PCR技术的基本构成、测量原理,以及实时荧光定量PCR仪的部件构成、质量控制和影响其检测精密度的因素。

  4. Establishment of a minor groove binder-probe based quantitative real time PCR to detect Borrelia burgdorferi sensu lato and differentiation of Borrelia spielmanii by ospA-specific conventional PCR

    Directory of Open Access Journals (Sweden)

    Strube Christina

    2010-08-01

    Full Text Available Abstract Background Borrelia burgdorferi sensu lato (sl, the causative agent of Lyme borreliosis, is transmitted by ticks of the genus Ixodes as vector. For identification of Borrelia infections in ticks a TaqMan™ minor groove binder (MGB probe-based quantitative real time PCR (qPCR was established targeting the 5S-23S intergenic spacer. Extension to a duplex qPCR included an Ixodes spp. positive control to verify successful DNA isolation. Besides qPCR, an ospA-specific conventional PCR for species-specific identification of B. spielmanii was established. Afterwards 1000 I. ricinus flagged in the city of Hanover, Germany, were investigated for B. burgdorferi sl infections followed by species identification. Furthermore, I. hexagonus ticks were investigated to proof applicability of the PCRs. Results Quantitative real time PCR (qPCR identifying B. burgdorferi sl in ticks was able to detect 1-10 copies per reaction. B. spielmanii ospA-specific conventional PCR was also highly specific and showed no cross reactions with the other tested Borrelia species. From 1000 hanoveranian ticks 24.3% were positive compared to only 7.4% positives by dark-field microscopy. Related to tick stage 1.7% larvae, 18.1% nymphs, and 34.6% adults were positive. The most frequent species was B. garinii, followed by B. afzelii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss. 70.6% of I. ricinus were mono-infected, whereas 28.0% and 1.4% were infected with two and three Borrelia species, respectively. From 232 I. hexagonus collected from hedgehogs in different sites of Germany, qPCR detected 5.7% to be infected with B. burgdorferi sl, which were identified as B. afzelii, B. garinii and B. spielmanii. Conclusions The evaluated qPCR to detect B. burgdorferi sl in Ixodes spp. is highly specific and sensitive. As a duplex qPCR including detection of Ixodes spp. DNA it is the first DNA based technique incorporating a control for successful DNA isolation from

  5. Rapid quantification of viable Campylobacter on chicken carcasses by real-time PCR and propidium monoazide as a tool for quantitative risk assessment

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Hansen, Tina Beck

    2010-01-01

    of foodborne Campylobacter, combining real-time PCR (Q-PCR) with a simple propidium monoazide (PMA) sample treatment. In less than 3 hours, this method generates a signal from only viable and viable but non-culturable (VBNC) Campylobacter with an intact membrane. The method performance was evaluated...... by assessing the contribution to variability from individual chicken carcass rinse matrices, species of Campylobacter, and the efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with Ct......-values (R2 = 0.993), with a quantification range from 1×1021×107 CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R2 = 0.844). The amplification efficiency of the Q-PCR method was not affected by chicken rinse matrix...

  6. Rapid Quantification of Viable Campylobacter Bacteria on Chicken Carcasses, Using Real-Time PCR and Propidium Monoazide Treatment, as a Tool for Quantitative Risk Assessment

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Hansen, Tina Beck

    2010-01-01

    of foodborne Campylobacter, combining real-time PCR (Q-PCR) with a simple propidium monoazide (PMA) sample treatment. In less than 3 hours, this method generates a signal from only viable and viable but non-culturable (VBNC) Campylobacter with an intact membrane. The method performance was evaluated...... by assessing the contribution to variability from individual chicken carcass rinse matrices, species of Campylobacter, and the efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with Ct......-values (R(2) = 0.993), with a quantification range from 1x10(2)-1x10(7) CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R(2) = 0.844). The amplification efficiency of the Q-PCR method was not affected by chicken rinse...

  7. Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin ß subunit mRNAs to support endocrine disruptor research.

    Energy Technology Data Exchange (ETDEWEB)

    Villeneuve, Daniel L.; Miracle, Ann L.; Jensen, Kathleen M.; Degitz, Sigmund J.; Kahl, Michael D.; Korte, Joseph J.; Greene, Katie J.; Blake, Lindsey S.; Linnum, Ann; Ankley, Gerald T.

    2007-03-01

    Fathead minnows (Pimephales promelas) are one of the most widely-used small fish models for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). In this study, we isolated and sequenced cDNAs for fathead minnow follicle-stimulating hormone-like and luteinizing hormone-like β (FSHβ and LHβ) and glycoprotein α (GPα) subunits. Quantitative real-time PCR assays for measuring gonadotropin (GtH) β subunit transcripts were developed and used to examine “baseline” transcript levels over a range of age classes and reproductive states encompassed in EDC testing. In females, FSHβ and LHβ transcripts were greater in 4-5 month old than in younger fish and were significantly correlated with one another across all age classes examined. In males, FSHβ transcripts were greatest in 2-3 month old fish and were inversely correlated with various measures of testis development including, gonadal-somatic index (GSI), and histological stage. Overall, the pattern of GtHβ expression over age classes associated with gonad development was similar to that reported for other asynchronous-spawning fish. Despite significant changes in female GSI, gonad stage, and plasma vitellogenin within 24 h of spawning, GtHβ transcript levels in fish that had spawned within the preceding 24 h were not significantly different from those in fish that were 2-3 days post-spawn and expected to spawn within the next 24 h based on spawning history. Results of this study provide insights related to the role of GtHs in fathead minnow reproductive development and function. Additionally they provide useful “baseline” data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression, which will facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain-pituitary-gonadal axis to stressors including EDCs.

  8. Correlation Between Pneumocystis jirovecii Mitochondrial Genotypes and High and Low Fungal Loads Assessed by Single Nucleotide Primer Extension Assay and Quantitative Real-Time PCR.

    Science.gov (United States)

    Alanio, Alexandre; Olivi, Martine; Cabaret, Odile; Foulet, Françoise; Bellanger, Anne-Pauline; Millon, Laurence; Berceanu, Ana; Cordonnier, Catherine; Costa, Jean-Marc; Bretagne, Stéphane

    2015-01-01

    We designed a single nucleotide primer extension (SNaPshot) assay for Pneumocystis jirovecii genotyping, targeting mt85 SNP of the mitochondrial large subunit ribosomal RNA locus, to improve minority allele detection. We then analyzed 133 consecutive bronchoalveolar lavage (BAL) fluids tested positive for P. jirovecii DNA by quantitative real-time PCR, obtained from two hospitals in different locations (Hospital 1 [n = 95] and Hospital 2 [n = 38]). We detected three different alleles, either singly (mt85C: 39.1%; mt85T: 24.1%; mt85A: 9.8%) or together (27%), and an association between P. jirovecii mt85 genotype and the patient's place of hospitalization (p = 0.011). The lowest fungal loads (median = 0.82 × 10(3) copies/μl; range: 15-11 × 10(3) ) were associated with mt85A and the highest (median = 1.4 × 10(6) copies/μl; range: 17 × 10(3) -1.3 × 10(7) ) with mt85CTA (p = 0.010). The ratios of the various alleles differed between the 36 mixed-genotype samples. In tests of serial BALs (median: 20 d; range 4-525) from six patients with mixed genotypes, allele ratio changes were observed five times and genotype replacement once. Therefore, allele ratio changes seem more frequent than genotype replacement when using a SNaPshot assay more sensitive for detecting minority alleles than Sanger sequencing. Moreover, because microscopy detects only high fungal loads, the selection of microscopy-positive samples may miss genotypes associated with low loads.

  9. Quantitative real-time reverse transcription-PCR analysis reveals stable and prolonged neurotoxin cluster gene activity in a Clostridium botulinum type E strain at refrigeration temperature.

    Science.gov (United States)

    Chen, Ying; Korkeala, Hannu; Lindén, Jere; Lindström, Miia

    2008-10-01

    The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30 degrees C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10 degrees C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30 degrees C. The maximum botE expression levels and average neurotoxin levels at 10 degrees C were 45 to 65% of those at 30 degrees C. The relative mRNA levels at 10 degrees C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30 degrees C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30 degrees C, whereas at 10 degrees C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.

  10. Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

    Directory of Open Access Journals (Sweden)

    Zhimin Yang

    Full Text Available Tall fescue (Festuca arundinacea Schreb. is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41 was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

  11. Selection of Reliable Reference Genes for Gene Expression Studies in the Biofuel Plant Jatropha curcas Using Real-Time Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Lu Zhang

    2013-12-01

    Full Text Available Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBasePLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha.

  12. Comparison of a quantitative real-time polymerase chain reaction (qPCR) with conventional PCR, bacterial culture and ELISA for detection of Mycobacterium avium subsp. paratuberculosis infection in sheep showing pathology of Johne's disease.

    Science.gov (United States)

    Sonawane, Ganesh G; Tripathi, Bhupendra N

    2013-12-01

    A quantitative real-time PCR (qPCR) assay employing IS900 gene specific primers of Mycobacterium avium subsp. parartuberculosis (MAP) was compared with conventional PCR, bacterial culture and enzyme-linked immunosorbent assay in 38 sheep showing granulomatous enteritis and lymphadenitis with and without demonstration of acid-fast bacilli (AFB). The lesions were classified as multibacillary (MB) (n = 23), which had diffuse granulomatous lesions with abundant AFB, and paucibacillary (PB) (n = 15), which had focal or multifocal granulomatous lesions with few or no AFB. In the multibacillary group (MB), IS900 PCR detected 19 (82.6%), and qPCR detected all 23 (100%) sheep positive for MAP in the intestine and lymph node tissues. In the paucibacillary group (PB), IS900 PCR detected 2 (13.3%), and qPCR detected all 15 (100%) sheep positive for MAP in tissues. When results of both groups were taken together, IS900 PCR detected 21(55.2%), and qPCR detected all 38 (100%) animals positive for MAP genome either in the intestine or lymph node tissues. On Herrold egg yolk medium, tissues of 14 (60.9%) MB and 5 (33.3%) PB sheep were found to be positive for MAP. Out of 27 sheep (PB = 8, MB = 19) tested by an ELISA, 21 (77.7%) were found to be positive for MAP antibody, of which 25% (2/8) and 100% (19/19) sheep were from PB and MB sheep, respectively. Based on the results of the present study, it was concluded that qPCR was a highly sensitive test in comparison to conventional PCR, ELISA and bacterial culture for the diagnosis of paratuberculosis on infected tissues especially from paucibacillary sheep.

  13. Quantitative detection and differentiation of free-living amoeba species using SYBR green-based real-time PCR melting curve analysis.

    Science.gov (United States)

    Behets, Jonas; Declerck, Priscilla; Delaedt, Yasmine; Verelst, Lieve; Ollevier, Frans

    2006-12-01

    Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.

  14. Calibration of quantitative real-time TaqMan PCR by correlation with hyphal biomass and ITS copies in mycelia of Piloderma croceum.

    Science.gov (United States)

    Raidl, S; Bonfigli, R; Agerer, R

    2005-11-01

    DNA-based quantification methods such as real-time TaqMan PCR allow a rapid and highly sensitive species-specific quantification of isolated fungal DNA material, but most quantification systems are only able to measure relative amounts of biomass or biomass changes during different treatments. In this experiment, an already established DNA quantification system for the ectomycorrhizal fungus Piloderma croceum, based on the ITS region of ribosomal DNA, was calibrated to absolute biomass to obtain a direct correlation between mycelial biomass and isolated ITS copies. Thin layers of sterile mycelia were cultured on slides. The mycelial biomass was calculated from measurements of the total hyphal length using image analysis, followed by determination of the mycelial volume, and multiplied by the specific weight of hyphae obtained from literature data. Using the very same mycelium, the number of ITS copies was quantified by TaqMan PCR. The mean value of 1047 (+/- 185) copies per mm hypha results in possible data for a direct conversion: one billion (10 (9)) ITS copies corresponded to 0.79 mg hyphal dry weight. For the ribosomal ITS multi-copy genes, the number of ITS copies could be calculated to approx. 152 (+/- 26) copies per dikaryotic cell. These conversion data now allow determination of the mycelial biomass of Piloderma croceum using real-time TaqMan PCR, a prerequisite for competition experiments with Piloderma croceum.

  15. Real-time PCR in Food Science: PCR Diagnostics.

    Science.gov (United States)

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

  16. Quantitative determination by real-time PCR of four vaginal Lactobacillus species, Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners

    Directory of Open Access Journals (Sweden)

    Tagg John R

    2007-12-01

    Full Text Available Abstract Background Most studies of the vaginal microflora have been based on culture or on qualitative molecular techniques. Here we applied existing real-time PCR formats for Lactobacillus crispatus, L. gasseri and Gardnerella vaginalis and developed new formats for Atopobium vaginae, L. iners and L. jensenii to obtain a quantitative non culture-based determination of these species in 71 vaginal samples from 32 pregnant and 28 non-pregnant women aged between 18 and 45 years. Results The 71 vaginal microflora samples of these women were categorized, using the Ison and Hay criteria, as refined by Verhelst et al. (2005, as follows: grade Ia: 8 samples, grade Iab: 10, grade Ib: 13, grade I-like: 10, grade II: 11, grade III: 12 and grade IV: 7. L. crispatus was found in all but 5 samples and was the most frequent Lactobacillus species detected. A significantly lower concentration of L. crispatus was found in grades II (p L. jensenii was found in all grades but showed higher concentration in grade Iab than in grade Ia (p = 0.024. A. vaginae and G. vaginalis were present in high concentrations in grade III, with log10 median concentrations (log10 MC, respectively of 9.0 and 9.2 cells/ml. Twenty (38.5% of the 52 G. vaginalis positive samples were also positive for A. vaginae. In grade II we found almost no L. iners (log10 MC: 0/ml but a high concentration of L. gasseri (log10 MC: 8.7/ml. By contrast, in grade III we found a high concentration of L. iners (log10 MC: 8.3/ml and a low concentration of L. gasseri (log10 MC: 0/ml. These results show a negative association between L. gasseri and L. iners (r = -0.397, p = 0.001 and between L. gasseri and A. vaginae (r = -0.408, p Conclusion In our study we found a clear negative association between L. iners and L. gasseri and between A. vaginae and L. gasseri. Our results do not provide support for the generally held proposition that grade II is an intermediate stage between grades I and III, because L

  17. Quantitative real-time PCR assay for rapid detection of plant and human pathogenic Macrophomina phaseolina from field and environmental samples.

    Science.gov (United States)

    Babu, Bandamaravuri Kishore; Mesapogu, Sukumar; Sharma, Anu; Somasani, Saida Reddy; Arora, Dilip K

    2011-01-01

    A real-time qPCR assay was developed to detect and quantify Macrophomina phaseolina abundance in rhizosphere soil and plant tissue. Both TaqMan and SYBR green techniques were targeted on ~ 1 kb sequence characterized amplified region (SCAR) of M. phaseolina and two sets of specific primers were designed for SYBR green (MpSyK) and TaqMan (MpTqK) assays. No cross-hybridization and no fluorescent signal exceeding the baseline threshold was observed in TaqMan and SYBR green assays, respectively. The minimum detection limit or sensitivity of TaqMan assay was 30 fg/μL of M. phaseolina DNA and limit of quantification of M. phaseolina viable population was estimated as 0.66 × 10(5) CFU/g soil(-1) equivalent to 10 pg/μL of target DNA. This is the first report which demonstrated real-time qPCR assays with greater specificity and sensitivity to detect M. phaseolina population in soil and plant materials.

  18. A simple method for normalization of DNA extraction to improve the quantitative detection of soil-borne plant pathogenic oomycetes by real-time PCR.

    Science.gov (United States)

    Li, M; Ishiguro, Y; Kageyama, K; Zhu, Z

    2015-08-01

    Most of the current research into the quantification of soil-borne pathogenic oomycetes lacks determination of DNA extraction efficiency, probably leading to an incorrect estimation of DNA quantity. In this study, we developed a convenient method by using a 100 bp artificially synthesized DNA sequence derived from the mitochondrion NADH dehydrogenase subunit 2 gene of Thunnus thynnus as a control to determine the DNA extraction efficiency. The control DNA was added to soils and then co-extracted along with soil genomic DNA. DNA extraction efficiency was determined by the control DNA. Two different DNA extraction methods were compared and evaluated using different types of soils, and the commercial kit was proved to give more consistent results. We used the control DNA combined with real-time PCR to quantify the oomycete DNAs from 12 naturally infested soils. Detectable target DNA concentrations were three to five times higher after normalization. Our tests also showed that the extraction efficiencies varied on a sample-to-sample basis and were DNA control for the normalization of DNA extraction by real-time PCR. By combining two different efficient soil DNA extraction methods, the developed quantification method dramatically improved the results. This study also proves that the developed normalization method is necessary and useful for the accurate quantification of soil-borne plant pathogenic oomycetes. © 2015 The Society for Applied Microbiology.

  19. Application of Real-time Fluorescence Quantitative PCR Technology in the Detection of Pathogens%实时荧光定量PCR技术在致病菌检测中的应用

    Institute of Scientific and Technical Information of China (English)

    周丽民; 雷永良; 王小光; 叶碧峰; 陈秀英

    2012-01-01

    目的 本文将实时荧光定量PCR (Real-time Q-PCR)法检查致病菌与细菌培养法作比较,探讨对指导临床诊断治疗的意义.方法 采用Real- time Q- PCR法和细菌培养法对100份标本进行6个微生物项目的检测,并比较两种方法的灵敏度、特异性和准确性.结果 Real-time Q-PCR法检测结果与细菌培养法,除完全符合的19例标本之外,另有8例用细菌培养法为阴性的标本,用Real- time Q- PCR法检测为阳性.结论 Real-time Q-PCR法较细菌培养法具有准确性好、灵敏度高、特异性强和检测快速等优点,是快速筛查致病菌的理想检测方法之一.%Objective To establish a rapid screening of pathogenic bacteria by the real- time quantitative PCR to guide the clinical diagnosis and treatment. Methods 100 specimens were tested by bacteria culture method in national standard and the real-time Q-PCR for six microbial items. The sensitivity, specificity and accuracy of the two methods were compared. Results The accordance rate of the real-time Q-PCR and the bacteria culture in 19 samples was100%. 8 specimens which showed negative with the traditional method were positive by realtime Q- PCR. Conclusion Real- time Q- PCR may be more accuracy, sensitivity, specific, and faster than that the bacteria culture. It may be used as an ideal methods in quick screening the pathogens of patients.

  20. Real-Time Quantitative PCR Technology and its Application in Detection of Infectious Diseases%实时荧光定量PCR及其在传染性疾病检测中的应用

    Institute of Scientific and Technical Information of China (English)

    邹红霞; 梁国鲁

    2011-01-01

    实时荧光定量PCR技术被广泛应用于实验研究、临床检测中.与普通的PCR相比,实时荧光定量PCR技术具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等优点.我们综述了实时荧光定量PCR技术的原理、定量方法,及其在传染性疾病检测研究中的应用.%In recent years, real-time quantitative PCR technology has been widely used in various experimental studies, clinical testing and production. Compared to normal PCR, real-time quantitative PCR technology is advanced in specificity, sensitivity, reproducibility, accuracy, speed and closed. This paper is a review about the principle, quantitative method of real-time fluorescence quantitative PCR technology, and the application in detection of infectious diseases.

  1. Efficient diagnosis and treatment follow-up of human brucellosis by a novel quantitative TaqMan real-time PCR assay: a human clinical survey.

    Science.gov (United States)

    Sohrabi, Majid; Mohabati Mobarez, Ashraf; Khoramabadi, Nima; Hosseini Doust, Reza; Behmanesh, Mehrdad

    2014-12-01

    Rapid and effective diagnosis of brucellosis is a challenge for clinicians. Even when diagnosis is on time and therapy is initiated, meticulous follow-up appointments are crucial for ensuring the efficacy of the treatment. Due to shortcomings of serological methods, molecular diagnosis, especially real-time PCR, is becoming a main approach in laboratory diagnostics. Thus, the development of efficient procedures and standardization of the PCR tests will have a great impact on the precise detection and quantification of bacterial DNA loads, which is valuable for the medical management of brucellosis patients. We developed a new TaqMan real-time PCR directed to bcsp31, a shared gene of the brucellae. The bcsp31 gene fragment was cloned into pJET1.2. Recombinant pJET1.2-bcsp31 was linearized by HindIII digestion, and the product was used for the preparation of a standard curve. A panel of Brucella spp. and non-Brucella pathogens was tested. No bacterial genomes other than those of the brucellae were detected. According to the results, specificity of the method was 100%. In a clinical assessment, the positive-control group comprised 37 patients with microbiologically confirmed brucellosis, and 25 healthy individuals served as the negative-control group. By the end of the treatment period, there was a significant decrease in the DNA load of the 37 brucellosis patients, which persisted for the 4 weeks of monitoring after treatment, suggesting that our proposed method is an efficient monitoring tool. Serum samples prior to any treatment were collected from the 25 serologically suspicious patients and assessed by our method; 72% of these patients tested positive for brucellosis.

  2. Lab-on-a-chip PCR: real time PCR in miniaturized format for HLA diagnostics

    Science.gov (United States)

    Gaertner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Sewart, René; Frank, Rainer; Willems, Andreas

    2014-05-01

    In case of transplantation or the identification of special metabolic diseases like coeliac disease, HLA typing has to be done fast and reliably with easy-to-handle devices by using limited amount of sample. Against this background a lab-on-a-chip device was realized enabling a fast HLA typing via miniaturized Real-time PCR. Hereby, two main process steps were combined, namely the extraction of DNA from whole blood and the amplification of the target DNA by Real-time PCR giving rise-to a semi-quantitative analysis. For the implementation of both processes on chip, a sample preparation and a real-time module were used. Sample preparation was carried out by using magnetic beads that were stored directly on chip as dry powder, together with all lysis reagents. After purification of the DNA by applying a special buffer regime, the sample DNA was transferred into the PCR module for amplification and detection. Coping with a massively increased surface-to-volume ratio, which results in a higher amount of unspecific binding on the chip surface, special additives needed to be integrated to compensate for this effect. Finally the overall procedure showed a sensitivity comparable to standard Real-time PCR but reduced the duration of analysis to significantly less than one hour. The presented work demonstrates that the combination of lab-on-a-chip PCR with direct optical read-out in a real-time fashion is an extremely promising tool for molecular diagnostics.

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2006-10-15

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

  5. Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus infections

    Directory of Open Access Journals (Sweden)

    Müller Marcel A

    2005-02-01

    Full Text Available Abstract Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.

  6. Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Vestergaard, Hanne; Møller, Michael Boe

    2011-01-01

    study, we present a real-time qPCR assay that allows quantification of as little as 0.003% KIT D816V mutation-positive cells. A total of 61 samples from 31 cases of SM were included in the study. We detected the mutation in skin or bone marrow in 95% of the cases of SM. We demonstrate the clinical...... relevance of the assay by identifying as little as 0.03% mutation-positive cells in bone marrow aspirates from SM patients and calculate the analytical sensitivity of negative samples to determine the reliability of the result. We further demonstrate that this method also detects the KIT D816V mutation...

  7. Modeling of Phenoxy Acid Herbicide Mineralization and Growth of Microbial Degraders in 15 Soils Monitored by Quantitative Real-Time PCR of the Functional tfdA Gene

    DEFF Research Database (Denmark)

    Bælum, Jacob; Prestat, Emmanuel; David, Maude M.

    2012-01-01

    continents. The mineralization patterns were fitted by zero/linear or exponential growth forms of the three-half-order models and by logarithmic (log), first-order, or zero-order kinetic models. Prior and subsequent to the mineralization event, tfdA genes were quantified using real-time PCR to estimate...... growth-linked kinetic models. A clear trend was observed that the mineralization rates of the three PAs occurred in the order 2,4-D > MCPA > MCPP, and a correlation was observed between rapid mineralization and soils exposed to PA previously. Finally, for 2,4-D mineralization, all seven mineralization...... patterns which were best fitted by the exponential model yielded a higher tfdA gene potential after mineralization had occurred than the three mineralization patterns best fitted by the Lin model....

  8. Detection and Quantification of Infectious Hypodermal and Hematopoietic Necrosis Virus and White Spot Virus in Shrimp Using Real-Time Quantitative PCR and SYBR Green Chemistry

    Science.gov (United States)

    Dhar, Arun K.; Roux, Michelle M.; Klimpel, Kurt R.

    2001-01-01

    A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the GeneAmp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (CT) values over a range of 1 to 105 copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp β-actin gene was amplified in parallel with the viral DNA. The CT values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard CT curves of these viruses. For each virus and its β-actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay. PMID:11474000

  9. A real-time semi-quantitative RT–PCR assay demonstrates that the pilE sequence dictates the frequency and characteristics of pilin antigenic variation in Neisseria gonorrhoeae

    OpenAIRE

    Rohrer, Melissa S.; Lazio, Matthew P.; Seifert, H. Steven

    2005-01-01

    A semi-quantitative real-time RT–PCR assay was designed to measure gonococcal pilin antigenicvariation (SQ-PCR Av assay). This assay employs 17 hybridization probe sets that quantitate subpopulations of pilin transcripts carrying different silent pilin copy sequences and one set that detects total pilE transcript levels. Mixtures of a DNA standard carrying the silent copy being detected and a clone encoding the starting pilE sequence, which is the majority pilE template, provided amplificatio...

  10. Quantitative Detection of Chlamydia psittaci and C. pecorum by High-Sensitivity Real-Time PCR Reveals High Prevalence of Vaginal Infection in Cattle

    OpenAIRE

    DeGraves, Fred J.; Gao, Dongya; Hehnen, Hans-Robert; Schlapp, Tobias; Kaltenboeck, Bernhard

    2003-01-01

    Bovine vaginal cytobrush specimens were analyzed for the presence of Chlamydia spp. by a high-sensitivity, high-specificity quantitative PCR. The 53% prevalence of low-level Chlamydia psittaci and C. pecorum genital infection detected in virgin heifers suggests predominantely extragenital transmission of Chlamydia in cattle and conforms to the high seroprevalence of anti-Chlamydia antibodies.

  11. Quantitative PCR - new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situhybridization

    DEFF Research Database (Denmark)

    Schlemmer, B.O.; Sørensen, B. S.; Overgaard, J.

    2004-01-01

    , and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real-time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained...... significant (p PCR compares with FISH and IHC. The data show...... that results obtained for amplification of HER2 by real-time PCR on the LightCycler instrument are comparable to results obtained by IHC and FISH....

  12. Detection of PRL-2 gene expression in hepatocellular carcinoma by real-time fluorescence quantitative PCR%实时荧光定量PCR检测原发性肝癌中PRL-2基因的表达

    Institute of Scientific and Technical Information of China (English)

    Chao Cheng; Amos Ela Bella; Ailin Guo; Guoyong Wu; Weikang Wu

    2009-01-01

    Objective: To quantitatively detect the expression level of PRL-2 in primary hepatocellular carcinoma using real-time fluorescence quantitative PCR. Methods: Total RNA isolated from human HCC and liver tissue adjacent to the tumor was reversely transcribed into cDNA. Real-time fluorescence quantitative PCR (Q-PCR) method was used to analyze the expres-sion level of PRL-2 gene. Results: The Q-PCR method was performed successfully to precisely detect RNA level. PRL-2 was expressed in all portal vein tumor thrombosis (PVTT) and HCC, but only in some paratumor tissue. The highest expression level of PRL-2 gene was recorded in PVTT; meanwhile expression level of PRL-2 was higher than that in paratumor liver tis-sues and in HCC (P<0.01), and it was higher in HCC than that in paratumor liver tissues. Conclusion: The Q-PCR may be the most precise method to quantitatively detect RNA level and can be used in gene expression changes. The PRL-2 gene has higher expression in PVTT than that in HCC and in paratumor liver tissue cells, indicating that it plays an important role in the development and metastasis of the HCC.

  13. Quantitation of viral load using real-time amplification techniques

    NARCIS (Netherlands)

    Niesters, H G

    2001-01-01

    Real-time PCR amplification techniques are currently used to determine the viral load in clinical samples for an increasing number of targets. Real-time PCR reduces the time necessary to generate results after amplification. In-house developed PCR and nucleic acid sequence-based amplification (NASBA

  14. Real-time PCR and PCR-tandem Mass Spectrometry for Biodetection

    Science.gov (United States)

    2005-10-01

    Real - time PCR and PCR- tandem mass spectrometry for biodetection Alvin Fox, University of South Carolina, School of Medicine Report Documentation...TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR and PCRtandem mass spectrometry for biodetection 5a. CONTRACT NUMBER 5b...interspace region Bacillus subtilis W23 standard Blank Barn dust House dust Cycle Real - time PCR (16s rRNA) - environmental samples Real - time

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xiaoping eNiu

    2015-10-01

    Full Text Available To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR, reliable reference gene(s are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as 3 different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions.

  17. Development, validation and field evaluation of a quantitative real-time PCR able to differentiate between field Mycoplasma synoviae and the MS-H-live vaccine strain.

    Science.gov (United States)

    Dijkman, R; Feberwee, A; Landman, W J M

    2017-08-01

    A quantitative PCR (qPCR) able to differentiate between field Mycoplasma synoviae and MS-H vaccine strain was developed, validated and evaluated. It was developed using nucleotide differences in the obg gene. Analytical specificity and sensitivity assessed using DNA from 194 M. synoviae field samples, three different batches of MS-H vaccine and from 43 samples representing four other avian Mycoplasma species proved to be 100%. The detection limit for field M. synoviae and MS-H vaccine strain was 10(2-3) and 10(2) colony-forming units PCR equivalents/g trachea mucus, respectively. The qPCR was able to detect both, field M. synoviae and MS-H vaccine strain in ratios of 1:100 determined both using spiked and field samples. One hundred and twenty samples from M. synoviae-infected non-vaccinated birds, 110 samples from M. synoviae-vaccinated birds from a bird experiment and 224 samples from M. synoviae negative (serology and PCR) birds were used to determine the relative sensitivity and specificity using a previously described M. synoviae PCR as reference. The relative sensitivity and specificity for field M. synoviae were 95.0% and 99.6%, respectively, and 94.6% and 100% for the MS-H-live vaccine, respectively. Field validation and confirmation by multi locus sequence typing revealed that the qPCR correctly distinguished between MS-H and field M. synoviae. Evaluation of the differentiating M. synoviae qPCR in three commercial flocks suggested transmission of MS-H-live vaccine from vaccinated to non-vaccinated flocks at the same farm. Furthermore, it showed evidence for the colonization with field M. synoviae in MS-H-vaccinated flocks.

  18. Overcoming inhibition in real-time diagnostic PCR.

    Science.gov (United States)

    Hedman, Johannes; Rådström, Peter

    2013-01-01

    PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency of PCR, thus lowering the detection limit, as well as the precision of sequence-specific nucleic acid quantification in real-time PCR. In order to overcome the problems caused by PCR inhibitors, all the steps leading up to DNA amplification must be optimized for the sample type in question. Sampling and sample treatment are key steps, but most of the methods currently in use were developed for conventional diagnostic methods and not for PCR. Therefore, there is a need for fast, simple, and robust sample preparation methods that take advantage of the accuracy of PCR. In addition, the thermostable DNA polymerases and buffer systems used in PCR are affected differently by inhibitors. During recent years, real-time PCR has developed considerably and is now widely used as a diagnostic tool. This technique has greatly improved the degree of automation and reduced the analysis time, but has also introduced a new set of PCR inhibitors, namely those affecting the fluorescence signal. The purpose of this chapter is to view the complexity of PCR inhibition from different angles, presenting both molecular explanations and practical ways of dealing with the problem. Although diagnostic PCR brings together scientists from different diagnostic fields, end-users have not fully exploited the potential of learning from each other. Here, we have collected knowledge from archeological analysis, clinical diagnostics, environmental analysis, food analysis, and forensic analysis. The concept of integrating sampling, sample treatment, and the chemistry of PCR, i.e., pre-PCR processing, will be addressed as a general approach to overcoming real-time PCR inhibition and producing samples optimal for PCR

  19. Modeling of Phenoxy Acid Herbicide Mineralization and Growth of Microbial Degraders in 15 Soils Monitored by Quantitative Real-Time PCR of the Functional tfdA Gene

    Science.gov (United States)

    Bælum, Jacob; Prestat, Emmanuel; David, Maude M.; Strobel, Bjarne W.

    2012-01-01

    Mineralization potentials, rates, and kinetics of the three phenoxy acid (PA) herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP), were investigated and compared in 15 soils collected from five continents. The mineralization patterns were fitted by zero/linear or exponential growth forms of the three-half-order models and by logarithmic (log), first-order, or zero-order kinetic models. Prior and subsequent to the mineralization event, tfdA genes were quantified using real-time PCR to estimate the genetic potential for degrading PA in the soils. In 25 of the 45 mineralization scenarios, ∼60% mineralization was observed within 118 days. Elevated concentrations of tfdA in the range 1 × 105 to 5 × 107 gene copies g−1 of soil were observed in soils where mineralization could be described by using growth-linked kinetic models. A clear trend was observed that the mineralization rates of the three PAs occurred in the order 2,4-D > MCPA > MCPP, and a correlation was observed between rapid mineralization and soils exposed to PA previously. Finally, for 2,4-D mineralization, all seven mineralization patterns which were best fitted by the exponential model yielded a higher tfdA gene potential after mineralization had occurred than the three mineralization patterns best fitted by the Lin model. PMID:22635998

  20. Detection of Live Salmonella sp. Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA▿ †

    OpenAIRE

    González-Escalona, Narjol; Hammack, Thomas S.; Russell, Mindi; Jacobson, Andrew P.; De Jesús, Antonio J.; Brown, Eric W.; Lampel, Keith A.

    2009-01-01

    Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponent...

  1. LUX real-time PCR assay for the detection of porcine circovirus type 2.

    Science.gov (United States)

    Vilcek, Stefan; Vlasakova, Michaela; Jackova, Anna

    2010-05-01

    Light Upon eXtension real-time PCR (LUX real-time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2 x 10(8) genome equivalents per assay with the best results in the range from 2 x 10(2) to 2 x 10(7) viral copies. The LUX real-time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between the LUX real-time PCR and the conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of the LUX real-time PCR with the TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real-time PCR, similar to the TaqMan PCR, was more specific for generation of fluorogenic signal than SYBR Green PCR.

  2. Establishment of Real-time PCR Assay for Quantitative of Plastid ACCase in Beardless Barnyardgrass%无芒稗质体ACCase基因Real-time PCR定量方法的建立

    Institute of Scientific and Technical Information of China (English)

    郇志博; 王金信

    2013-01-01

    为研究一种抗精喹禾灵无芒稗的抗药性机理,建立了一种以-actin为内参基因利用实时荧光定量PCR技术测定抗性无芒稗质体ACCase基因相对表达水平的方法.分析结果表明,设计的实时定量引物特异性非常好,内参基因、目的基因的扩增效率分别为103.2%、104.3%,线性相关系数分别为0.993 0、0.991 2,完全满足Real-time PCR定量方法的要求.所建立的无芒稗质体型ACCase基因Real-time PCR定量方法将为研究质体型ACCase基因的表达和杂草对ACCase抑制剂的抗药性机制研究提供方法学参考.

  3. Establishment of Real-time PCR Assay for Quantitative Analysis of AIHAK1%獐茅AIHAK1 Real-time PCR定量方法的建立

    Institute of Scientific and Technical Information of China (English)

    王兰; 苏乔

    2009-01-01

    以β-actin为内参基因,根据已克隆的AIHAK1及β-actin碱基序列,分别设计了实时定量特异性引物,优化了反应的退火温度与引物浓度,并以优化的条件建立了相对定量标准曲线,同时对该方法的稳定性进行了评价.结果表明AIHAK1及β-actin基因的real-time PCR扩增效率分别为1.09和1.04,线性相关系数分别为0.996和0.997,批内及批间变异系数<5%.所建立的A1HAK1 real-time PCR定量方法操作简便、定量准确、重复性好,为进一步探索AIHAK1的功能、mRNA表达水平的变化及转AIHAK1农作物的检测奠定了方法学基础.

  4. 罗非鱼组织内无乳链球菌实时荧光定量 PCR 检测方法建立%Real-time quantitative PCR for detection of Streptococcus agalactiae from tilaPia tissue

    Institute of Scientific and Technical Information of China (English)

    王瑞; 李莉萍; 黄婷; 梁万文; 梁聪; 雷爱莹; 陈明

    2015-01-01

    . The results show that the real-time quantitative PCR assay which we developed was accurate,specific and sen-sitive,and could detect S. agalactiae from various tilapia tissues rapidly. The method can be used for surveillance and prevention of S. agalactiae disease of tilapia.

  5. Detection of Tumor Markers in Prostate Cancer and Comparison of Sensitivity between Real Time and Nested PCR

    OpenAIRE

    Matsuoka, Takayuki; Shigemura, Katsumi; Yamamichi, Fukashi; Fujisawa, Masato; Kawabata, Masato; Shirakawa, Toshiro

    2012-01-01

    The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivi...

  6. Detection of Tumor Markers in Prostate Cancer and Comparison of Sensitivity between Real Time and Nested PCR

    OpenAIRE

    Matsuoka, Takayuki; Shigemura, Katsumi; Yamamichi, Fukashi; Fujisawa, Masato; Kawabata, Masato; Shirakawa, Toshiro

    2012-01-01

    The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivi...

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

    Science.gov (United States)

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

    2002-01-01

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

  8. Development of a multiplex Real-time fluorescent quantitative PCR assay for simultaneous detection of PRV,PPV and PCV2%3种猪繁殖障碍性病毒Real-time PCR快速检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    赵绪永; 马辉; 宁豫昌; 赵丽

    2012-01-01

    【目的】建立可同时检测猪伪狂犬病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒Ⅱ型(PCV2)的多重实时荧光定量PCR方法。【方法】根据GenBank数据库中PRV、PPV和PCV2的核苷酸序列,设计3对特异性引物和探针,以10倍系列稀释的阳性质粒为模板,优化反应条件,建立检测PRV、PPV和PCV2的多重Real-time PCR方法,并对其敏感性、重复性和特异性进行检验;分别采用单项和多重Real-time PCR方法,对临床收集的42份疑似病料进行检测,比较2种方法的符合率。【结果】特异性和灵敏度试验表明,建立的多重Real-time PCR检测方法具有高度特异性,与其他病原无明显交叉反应;检测灵敏度高,可检出1.0×101拷贝/μL的阳性质粒或1TCID50/mL的病毒样品。用多重Real-time PCR对42份临床疑似病料进行检测,其检测结果与单重Real-time PCR结果完全一致,表明多重Real-time PCR方法是可行的。【结论】建立了可同时检测PRV、PPV和PCV2的多重Real-time PCR方法,该法具有快速、灵敏、特异和重复性好等优点。%【Objective】 The study developed a multiplex real-time fluorescent quantitative PCR which can simultaneously detect and discriminate porcine pseudorabies virus(PRV),porcine parvovirus(PPV) and porcine circovirus type 2(PCV2).【Method】 According to the nucleotide sequences of PRV,PPV and PCV2 from GenBank,3 pairs of specific primers and probes were designed.The positive plasmid diluted by 10 times was used as template to establish a multiplex Real-time PCR assay by optimizing the reaction conditions.Sensitivity,reproducibility and specificity assays were determined.42 clinical suspected disease materials were detected by the established multiplex Real-time PCR assay and compared with the result of singleplex assay.【Result】 The results of specificity and sensitivity assays showed that the specificity of the established multiplex Real-time PCR assay was high

  9. Shape based kinetic outlier detection in real-time PCR

    Directory of Open Access Journals (Sweden)

    D'Atri Mario

    2010-04-01

    Full Text Available Abstract Background Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. Results Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific. Conclusion Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.

  10. Selection and validation of reference genes for quantitative real-time PCR in buckwheat (Fagopyrum esculentum) based on transcriptome sequence data.

    Science.gov (United States)

    Demidenko, Natalia V; Logacheva, Maria D; Penin, Aleksey A

    2011-05-12

    Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits). These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications--geNorm, NormFinder and BestKeeper--were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1), AT2G28390 (SAND family protein, SAND) and AT5G46630 (clathrin adapter complex subunit family protein, CACS) are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species.

  11. Selection and validation of reference genes for quantitative real-time PCR in buckwheat (Fagopyrum esculentum based on transcriptome sequence data.

    Directory of Open Access Journals (Sweden)

    Natalia V Demidenko

    Full Text Available Quantitative reverse transcription PCR (qRT-PCR is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits. These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications--geNorm, NormFinder and BestKeeper--were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1, AT2G28390 (SAND family protein, SAND and AT5G46630 (clathrin adapter complex subunit family protein, CACS are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species.

  12. Introducing Undergraduate Students to Real-Time PCR

    Science.gov (United States)

    Hancock, Dale; Funnell, Alister; Jack, Briony; Johnston, Jill

    2010-01-01

    An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis,…

  13. Real-Time PCR Quantification of Methanobrevibacter oralis in Periodontitis

    OpenAIRE

    Bringuier, Amélie; Khelaifia, Saber; Richet, Hervé; Aboudharam, Gérard; Drancourt, Michel

    2013-01-01

    A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P = 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P = 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis.

  14. Introducing Undergraduate Students to Real-Time PCR

    Science.gov (United States)

    Hancock, Dale; Funnell, Alister; Jack, Briony; Johnston, Jill

    2010-01-01

    An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis,…

  15. Real-time PCR for Strongyloides stercoralis-associated meningitis.

    Science.gov (United States)

    Nadir, Eyal; Grossman, Tamar; Ciobotaro, Pnina; Attali, Malka; Barkan, Daniel; Bardenstein, Rita; Zimhony, Oren

    2016-03-01

    Four immunocompromised patients, immigrants from Ethiopia, presented with diverse clinical manifestations of meningitis associated with Strongyloides stercoralis dissemination as determined by identification of intestinal larvae. The cerebrospinal fluid of 3 patients was tested by a validated (for stool) real-time PCR for S. stercoralis and was found positive, establishing this association.

  16. Towards a portable microchip system with integrated thermal control and polymer waveguides for real-time PCR

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Sekulovic, Andrea; Kutter, Jörg Peter

    2006-01-01

    performed with a dilution series of C. jejuni DNA template (2 to 200 pg/mu l) could be quantitatively detected and compared with a conventional post-PCR analysis (DNA gel electrophoresis). The presented approach provided reliable real-time quantitative information of the PCR amplification of the targeted......A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. The integrated polymer optical system for real-time monitoring of PCR was fabricated in the same SU-8 layer as the PCR chamber, without additional masking steps. Two suitable DNA...... binding dyes, SYTOX Orange and TO-PRO-3, were selected and tested for the real-time PCR processes. As a model, cadF gene of Campylobacter jejuni has been amplified on the microchip. Using the integrated optical system of the real-time PCR microchip, the measured cycle threshold values of the real-time PCR...

  17. Molecular sensitivity threshold of wet mount and an immunochromatographic assay evaluated by quantitative real-time PCR for diagnosis of Trichomonas vaginalis infection in a low-risk population of childbearing women.

    Science.gov (United States)

    Leli, Christian; Castronari, Roberto; Levorato, Lucia; Luciano, Eugenio; Pistoni, Eleonora; Perito, Stefano; Bozza, Silvia; Mencacci, Antonella

    2016-06-01

    Vaginal trichomoniasis is a sexually transmitted infection caused by Trichomonas vaginalis, a flagellated protozoan. Diagnosis of T. vaginalis infection is mainly performed by wet mount microscopy, with a sensitivity ranging from 38% to 82%, compared to culture, still considered the gold standard. Commercial immunochromatographic tests for monoclonal-antibody-based detection have been introduced as alternative methods for diagnosis of T. vaginalis infection and have been reported in some studies to be more sensitive than wet mount. Real-time PCR methods have been recently developed, with optimal sensitivity and specificity. The aim of this study was to evaluate whether there is a molecular sensitivity threshold for both wet mount and imunochromatographic assays. To this aim, a total of 1487 low-risk childbearing women (median age 32 years, interquartile range 27-37) were included in the study, and underwent vaginal swab for T. vaginalis detection by means of a quantitative real-time PCR assay, wet mount and an immunochromatographic test. Upon comparing the results, prevalence values observed were 1.3% for real-time PCR, 0.5% for microscopic examination, and 0.8% for the immunochromatographic test. Compared to real-time PCR, wet mount sensitivity was 40% (95% confidence interval 19.1% to 63.9%) and specificity was 100% (95% CI 99.7% to 100%). The sensitivity and specificity of the immunochromatographic assay were 57.9% (95% CI 33.5% to 79.8%) and 99.9% (95% CI 99.6% to 100%), respectively. Evaluation of the wet mount results and those of immunochromatographic assay detection in relation to the number of T. vaginalis DNA copies detected in vaginal samples showed that the lower identification threshold for both wet mount (chi-square 6.1; P = 0.016) and the immunochromatographic assay (chi-square 10.7; P = 0.002) was ≥100 copies of T. vaginalis DNA/5 mcl of eluted DNA.

  18. Application of real-time PCR for quantitative detection of Campylobacter jejuni by TaqMan probe%基于TaqMan探针的Real-time PCR定量检测空肠弯曲杆菌

    Institute of Scientific and Technical Information of China (English)

    阳成波; 蒋原; 黄克和; 祝长青

    2003-01-01

    空肠弯曲杆菌(Campylobacter jejuni,C.jijuni)被认为是人类主要食源性病原菌之一.由于空肠弯曲杆菌特殊的生长条件和容易进入不可培养但存活的状态(Viable but nonculturable,VNC),所以传统的生化鉴定结果并不一定可靠,并且是一项费时而繁琐的工作.核酸检测方法的出现为空肠弯曲杆菌的检测带来了方便.在本文基于Light Cycler为平台,建立一种基于TaqMan探针的Real-time PCR方法来定量检测空肠弯曲杆菌.用该方法检测时,发现所有空肠弯曲杆菌(11株)都呈阳性,所有其它弯曲菌(3株)和其它菌株(5株)都是阴性.整个检测过程60 min内可以完成,检测限度为5 CFU,标准曲线的相关系数为0.988.结果表明荧光定量PCR方法既为空肠弯曲杆菌提供了一种特异、敏感、快速和简洁的定量检测方法,又为研究空肠弯曲杆菌致病机理提供了一种重要方法.

  19. Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes

    Directory of Open Access Journals (Sweden)

    Jonathan W.K. Liew

    2017-07-01

    Full Text Available Quantitative reverse transcription PCR (qRT-PCR has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion. Two anti-Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1 and nitric oxide synthase (NOS, play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. However, these are less studied in Anopheles dirus, a dominant malaria vector in Southeast Asia. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. This may lead to erroneous quantification of expression levels. Therefore, the present study characterized and investigated the expression profiles of TEP1 and NOS of Anopheles dirus during P. berghei infection. Prior to that, the elongation factor 1-alpha (EF1, actin 1 (Act and ribosomal protein S7 (S7 genes were validated for their suitability as a set of reference genes. TEP1 and NOS expressions in An. dirus were found to be significantly induced after P. berghei infection.

  20. Overestimation of the Legionella spp. load in environmental samples by quantitative real-time PCR: pretreatment with propidium monoazide as a tool for the assessment of an association between Legionella concentration and sanitary risk.

    Science.gov (United States)

    Ditommaso, Savina; Ricciardi, Elisa; Giacomuzzi, Monica; Arauco Rivera, Susan R; Ceccarelli, Adriano; Zotti, Carla M

    2014-12-01

    Quantitative polymerase chain reaction (qPCR) offers rapid, sensitive, and specific detection of Legionella in environmental water samples. In this study, qPCR and qPCR combined with propidium monoazide (PMA-qPCR) were both applied to hot-water system samples and compared to traditional culture techniques. In addition, we evaluated the ability of PMA-qPCR to monitor the efficacy of different disinfection strategies. Comparison between the quantification obtained by culture and by qPCR or PMA-qPCR on environmental water samples confirms that the concentration of Legionella estimated by GU/L is generally higher than that estimated in CFU/L. Our results on 57 hot-water-system samples collected from 3 different sites show that: i) qPCR results were on average 178-fold higher than the culture results (Δ log10=2.25), ii) PMA-qPCR results were on average 27-fold higher than the culture results (Δ log10=1.43), iii) propidium monoazide-induced signal reduction in qPCR were nearly 10-fold (Δ log10=0.95), and that iv) different degrees of correlations between the 3 methods might be explained by different matrix properties, but also by different disinfection methods affecting cultivability of Legionella. In our study, we calculated the logarithmic differences between the results obtained by PMA-qPCR and those obtained by culture, and we suggested an algorithm for the interpretation of PMA-qPCR results for the routine monitoring of healthcare water systems using a commercial qPCR system (iQ-check real-time PCR kit; Bio-Rad, Marnes-la-Coquette, France).

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  2. Real-time fluorescence quantitative PCR detecting legionella in sputum specimens%实时荧光定量PCR检测痰标本中军团菌属

    Institute of Scientific and Technical Information of China (English)

    黄宪章; 朱庆义; 周华友; 唐小龙; 熊玉娟; 胡朝晖; 刘洋敏; 宣瑞红; 赵利伟; 李连青

    2011-01-01

    OBJECTIVE To establish a fluorescent quantitative PCR method of Legionella-specific 16S rRNA gene,and to investigate the patients with pneumonia of Legionella infection in Guangzhou. METHODS 16S rRNA gene of Legionella spp. was used to design primers and probes. The reaction system and reaction conditions were optimized and the specificity, sensitivity and repeatability of this method were verified by detecting Legionella pneumophila, non-Legionella pneumophila and other bacteria. Totally 532 sputum specimens of patients with pneumonia in Guangdong Provincial Hospital of Chinese Medicine were detected. RESULTS The results showed that all reference strains of Legionella were positive, while all of other bacteria were negative, and the sensitivity was 10 CFU/ml. The positive rate of fluorescent quantitative PCR was 8.08% sputum specimens, while the 16S rRNA PCR and gene sequencing was 7.71% (41/532) positive. This results showed that no significant difference between the two groups, which had good compliance and equivalence. CONCLUSION 16S rRNA gene fluorescent quantitative PCR method can detect Legionella, it is fast, sensitive and specified and suitable for clinical investigation of legionella infection and the rapid detection.%目的:建立荧光定量PCR方法,用于检测军团菌属特异性16S rRNA基因,并探讨广州地区军团菌属感染状况.方法:利用军团菌属特异性16S rRNA基因保守序列设计引物和探针,优化反应条件和反应体系,对嗜肺军团菌、非嗜肺军团菌及其他细菌进行检测,验证该方法的特异性、敏感性、重复性,对广东省中医院采集的532例肺部感染患者痰液标本进行检测.结果:该方法检测军团菌属所有标准菌株均出现阳性信号,其他非军团菌属细菌检测结果均为阴性;检测灵敏度为102CFU/ml,临床检测532例肺部感染患者的痰液标本,荧光法检出军团菌阳性43例,阳性率为8.08%,16S rRNA PCR法加基

  3. Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance

    Directory of Open Access Journals (Sweden)

    Ralevski Filip

    2009-12-01

    Full Text Available Abstract Background Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. Materials and methods A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR, and two rapid diagnostic immuno-chromatographic tests (ICT in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. Results QPCR is the most analytically sensitive method (sensitivity 99.41%, followed by CARESTART (sensitivity 88.24%, and BINAXNOW (sensitivity 86.47% for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R2 = 0.9746 in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/μl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more

  4. 基孔肯雅病毒的纳米金实时荧光PCR方法的建立%Development of gold nanoparticle-assisted real-time fluorescence PCR for quantitative detection of Chikungunya viruses

    Institute of Scientific and Technical Information of China (English)

    燕清丽; 杨鹏飞; 房健慧; 张丽萍; 张晓龙; 曹晓梅; 姚李四

    2012-01-01

    目的 建立一种检测基孔肯雅病毒纳米金实时荧光定量PCR的方法.方法 以课题组建立的普通实时荧光PCR方法为基础,在PCR体系中添加不同大小粒径的纳米金进行体系优化,评价优化后的体系.结果 添加纳米金的实时荧光PCR较不添加的扩增效率高;优化后的纳米金实时荧光PCR产物的Ct值与模板稀释浓度存在良好的线性关系,回归方程:y=-3.31x+41.78,R2=0.9997,PCR扩增效率为99.5%.结论 纳米金能提高实时荧光PCR的反应效率.%Objective To develop a gold nanoparticle-assisted real-time fluorescence PCR array for quantitative detection of Chikungunya viruses. Methods On the basis of the real-time PCR that we had established, gold nanoparticles of different sizes were added to the system for optimization, which was then evaluated. Results It was found that the amplification efficacy of the system with gold nanoparticles was higher than that without gold nanoparticles, amounting to 99.5%. There existed a good linear relationship between the Ct value of the products by gold nanoparticle-assisted real-time fluorescence PCR and the concentrations of templates, with the regression equation being y=-3.31x+ 41.78,R2=0.9997. Conclusion Gold nanoparticle-assisted realtime fluorescence PCR has the potential to improve the efficiency of PCR amplification.

  5. Sequence polymorphism can produce serious artifacts in real-time PCR assays: lessons from Pacific oysters

    Science.gov (United States)

    Since it was first described in the mid-1990s, quantitative real time PCR (Q-PCR) has been widely used in many fields of biomedical research and molecular diagnostics. This method is routinely used to validate whole transcriptome analyses such as DNA microarrays, suppressive subtractive hybridizati...

  6. Interlaboratory Comparison of Real-time PCR Protocols for Quantification of General Fecal Indicator Bacteria

    Science.gov (United States)

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

  7. 实时荧光定量PCR技术及其在生命科学领域中的应用(二)%Real-time Fluorescent Quantitative PCR Technique and its Application in the Field of Life Science

    Institute of Scientific and Technical Information of China (English)

    袁媛; 陈强; 陈思祗; 刘中来

    2006-01-01

    实时荧光定量PCR(real-time fluorescent quantitative PCR,FQ-PCR)技术是在PCR定性技术基础上诞生起来的用不同荧光定量核酸的技术,具有敏感性高、重复性好、速度快、操作简便和污染少等优点.本文综述了FQ-PCR技术的原理、特点、目前常用FQ-PCR仪、定量方法、实验条件及实验中的主要影响因素及FQ-PCR技术在生命科学领域中的应用和研究进展.

  8. Real-time Fluorescent Quantitative PCR Technique and its Application in the Field of Life Science%实时荧光定量PCR技术及其在生命科学领域中的应用(一)

    Institute of Scientific and Technical Information of China (English)

    袁媛; 陈强; 陈思祗; 刘中来

    2006-01-01

    实时荧光定量PCR(real-time fluorescent quantitative PCR,FQ-PCR)技术是在PCR定性技术基础上发展起来的用不同荧光定量核酸的技术,具有敏感性高、重复性好、速度快、操作简便和污染少等优点.本文综述FQ-PCR技术的原理、特点、目前常用FQ-PCR仪、定量方法、实验条件及实验中的主要影响因素及FQ-PCR技术在生命科学领域中的应用和研究进展.

  9. Development of a novel multiplex type-specific quantitative real-time PCR for detection and differentiation of infections with human papillomavirus types HPV2, HPV27, and HPV57.

    Science.gov (United States)

    Hošnjak, Lea; Fujs Komloš, Kristina; Kocjan, Boštjan J; Seme, Katja; Poljak, Mario

    2016-12-01

    The present study describes the development and evaluation of the first multiplex type-specific quantitative real-time PCR (RT-PCR), enabling simple, rapid, sensitive, and specific concurrent detection and differentiation of human papillomavirus (HPV) types HPV2, 27, and 57 in a single PCR reaction. The HPV2/27/57 multiplex RT-PCR with a dynamic range of seven orders of magnitude (discriminating 10 to 108 viral genome equivalents/reaction) has an analytical sensitivity of at least 10 viral copies of each targeted HPV type/reaction, and no cross-reactivities were observed among the included targets. All three primer/probe combinations were efficient in amplifying 500 copies of targeted DNA in a background of 108, 107, 500, 100, and 10 copies of non-targeted viral DNA/reaction, and the performance of the HPV2/27/57 multiplex RT-PCR was additionally not affected by the presence of background human genomic DNA. When testing DNA isolates obtained from fresh-frozen tissue specimens of various children's warts, the results of the HPV2/27/57 multiplex RT-PCR were completely in line with the results of the conventional Low-risk Alpha-PV PCR. The newly developed HPV2/27/57 multiplex RT-PCR is an appropriate test for use in routine clinical laboratory settings and for studies focusing on the molecular epidemiology, pathogenesis, and natural history of HPV2/27/57-related lesions.

  10. Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR

    Directory of Open Access Journals (Sweden)

    Wenlong Bao

    2016-07-01

    Full Text Available Cunninghamia lanceolata (Chinese fir is a fast-growing and commercially important conifer of the Cupressaceae family. Due to the unavailability of complete genome sequences and relatively poor genetic background information of the Chinese fir, it is necessary to identify and analyze the expression levels of suitable housekeeping genes (HKGs as internal reference for precise analysis. Based on the results of database analysis and transcriptome sequencing, we have chosen five candidate HKGs (Actin, GAPDH, EF1a, 18S rRNA, and UBQ with conservative sequences in the Chinese fir and related species for quantitative analysis. The expression levels of these HKGs in roots and cotyledons under five different abiotic stresses in different time intervals were measured by qRT-PCR. The data were statistically analyzed using the following algorithms: NormFinder, BestKeeper, and geNorm. Finally, RankAggreg was applied to merge the sequences generated from three programs and rank these according to consensus sequences. The expression levels of these HKGs showed variable stabilities under different abiotic stresses. Among these, Actin was the most stable internal control in root, and GAPDH was the most stable housekeeping gene in cotyledon. We have also described an experimental procedure for selecting HKGs based on the de novo sequencing database of other non-model plants.

  11. A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

    Science.gov (United States)

    McFall, Sally M; Wagner, Robin L; Jangam, Sujit R; Yamada, Douglas H; Hardie, Diana; Kelso, David M

    2015-03-01

    Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.

  12. Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

    Directory of Open Access Journals (Sweden)

    Reis Patricia P

    2010-06-01

    Full Text Available Abstract Background MicroRNAs (miRs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE tissue. Results Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p 35, we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p Conclusion Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

  13. TaqMan MGB probe real- time fluorescence quantitative PCR for rapid detection of Mycoplasma%TaqMan MGB探针法实时荧光定量PCR快速检测支原体的研究

    Institute of Scientific and Technical Information of China (English)

    高正琴; 邢进; 冯育芳; 岳秉飞; 贺争鸣

    2011-01-01

    目的:建立特异、敏感、快速检测支原体的TaqMan MGB探针实时荧光定量PCR方法.方法:针对支原体16S rRNA基因的保守区设计特异性引物和探针,建立支原体TaqMan MGB探针实时荧光定量PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008~2010年期间在北京采集的680份小型猪、小鼠、大鼠样本中的支原体进行检测,同时进行分离培养和常规PCR检测.结果:建立的TaqMan MGB探针实时荧光定量PCR方法对支原体的检测具有高度的特异性,对空肠弯曲菌、支气管鲍特杆菌、肺炎克雷伯杆菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌、肺炎链球菌、乙型溶血性链球菌均无交叉反应,检测的灵敏度达9.2拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.328,TaqMan MGB探针实时荧光定量PCR效率为100%.对680份动物样本进行检测,结果TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出77份支原体阳性样本,但分离培养未能检出支原体阳性样本.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从动物样本中检出支原体DNA,检测时间仅为2h.结论:本研究建立了一种可靠、快速、灵敏的检测支原体的TaqMan MGB探针实时荧光定量PCR方法,并且成功应用于小型猪、小鼠、大鼠样本中支原体的检测.该技术为动物源性药品和生物制品中支原体的快速检测提供了实用的工具.%Objective: To develop a TaqMan MGB probe - based, sensitive and specific real - time fluorescence quantitative PCR assay for rapid detection of Mycoplasma. Methods: Primers and probes specific to 16S rRNA gene of Mycoplasma were designed. A TaqMan MGB probe - based, real - time fluorescence quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed. Then, the established TaqMan MGB

  14. Employing Real Time PCR for the Diagnosis of Huntington Disease

    Directory of Open Access Journals (Sweden)

    Frouzandeh Mahjoubi

    2013-07-01

    Full Text Available Background: Huntington disease (HD is a dominantly inherited, neurodegenerative disease characterized by choreiform movement disturbances and dementia. The onset age of this disease is varied but usually is between the ages 40-50. Huntington's disease is caused by a triplet-repeat expansion in the IT15 gene (also known as huntingtin or HD which is located on chromosome 4p3.1. Since many clinical picture of HD are indistinguishable from other distinct genetic disorders molecular test such as PCR is the only way to confirm the disease. The aim of this study was to introduce a new and fast technique for the diagnosis of Huntington disease.Materials and Methods: Blood specimens were collected from individuals suspected for Huntington disease and also people with no symptoms and family history of this disease. DNAs were extracted according to standard protocol. Using conventional PCR, patient positive for Huntington disease were diagnosed. Then employing real time PCR on the basis of difference between melting temperature (Tm a new and fast diagnostic method was introduced.Results: Among 29 patients suspected to be HD only 8 HD patients were confirmed using PCR and real time PCR. The numbers of CAG repeat were between 42-50 and melting temperatures were between 89-92.Conclusion: The concept of using melting temperature in real time PCR protocol presented in here could be employed for the rapid diagnosis of the diseases caused by the increased in triple repeat sequences. It is fast, robust and has the potential use for the prenatal diagnosis.

  15. 实时荧光定量PCR技术在食源性致病菌检测中的应用%Application of Real-time Fluorescent Quantitative PCR for the Detection of Food-borne Pathogenic Bacteria

    Institute of Scientific and Technical Information of China (English)

    吕艳芳; 马春颖; 励建荣

    2014-01-01

    近年来食源性疾病爆发数量呈上升趋势,食品安全问题已经成为危害人们生命和健康的严重问题,引起了全世界消费者对食品品质、安全和卫生的关注。实时荧光定量PCR技术是在定性PCR技术的基础上发展起来的核酸定量技术,灵敏性高、特异性强,实现了对DNA模板的定量检测,目前已广泛应用于食品检测领域。本文概述了实时荧光定量PCR技术的原理及其在食源性致病菌检测中的应用,并对其应用前景进行了探讨。%The outbreak of food-borne disease has shown a rising trend in recent years, and the problem of food safety seriously threats people’s life and health. Consumers around the world pay much attention to food quality, safety and health. Real-time fluorescent quantitative PCR technique is a nucleic acid quantitative technique, on the basis of the qualitative PCR technology, having high sensitivity, strong specificity, which has realized the quantita-tive detection of DNA template, and now has been widely used in the field of food detection. This paper summa-rized the principle of real-time fluorescent quantitative PCR technique and its application in the detection of food-borne pathogenic bacteria, and discussed the application prospect.

  16. Result Variation and Efficiency Kinetics in Real-Time PCR

    Directory of Open Access Journals (Sweden)

    Reza Shahsiah

    2010-10-01

    Full Text Available Fluorescent monitoring of DNA amplification is the basis of real-time PCR. Absolute quantification can be achieved using a standard curve method. The standard curve is constructed by amplifying known amounts of standards under identical conditions to that of the samples.The objective of the current study is to propose a mathematical model to assess the acceptability of PCR resulys.Four commercial standards for HCV-RNA (hepatitis C virus RNA along with 6 patient samples were measured by real-time PCR, using two different RT-PCR reagents. The standard deviation of regression (Sy,x was calculated for each group of standard and compared by F-Test. The efficiency kinetics was computed by logistic regression, c2 goodness of fit test was preformed to assess the appropriateness of the efficiency curves.Calculated efficiencies were not significantly different from the value predicted by logistic regression model. Reactions with more variation showed less stable efficiency curves, with wider range of amplification efficiencies.Amplification efficiency kinetics can be computed by fitting a logistic regression curve to the gathered fluorescent data of each reaction. This model can be employed to assess the acceptability of PCR results calculated by standard curve method.

  17. Development of a real-time PCR method for Thalassiosira rotula rapid detection

    Institute of Scientific and Technical Information of China (English)

    HE Shanying; YU Zhigang; MI Tiezhu

    2007-01-01

    Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ - PCR) method was developed for quantitative detection of T. rotula. The RFQ - PCR assay data showed that the results obtained with the RFQ - PCR quite good agreement with those with the light microscope (LM) counting method, which suggested that the RFQ - PCR could be a useful method for red tide alga detection.

  18. Simultaneous detection of West Nile virus and Chikungunya virus by duplex real-time quantitative PCR%双重荧光定量PCR检测西尼罗病毒和基孔肯雅病毒

    Institute of Scientific and Technical Information of China (English)

    余蓓蓓; 卢亦愚; 谢鑫友; 徐昌平; 张钧

    2013-01-01

    目的 建立同时检测西尼罗病毒(WNV)、基孔肯雅病毒(CHIKV)的双重荧光定量PCR法,为临床疑似病例的诊断提供依据.方法 分别针对WNV CAP基因、CHIKV E1基因保守区设计特异性引物和TaqMan探针,建立并优化双重荧光定量PCR反应体系,评价方法的特异性和灵敏度.结果 建立的双重荧光定量PCR可同时检测WNV、CHIKV核酸,标准曲线相关系数(r)分别达0.999、0.998,灵敏度达10 copies/μL,具有良好的特异性.结论 建立了同时检测WNV、CHIKV的双重荧光定量PCR法,但尚需临床进一步验证.%Objective To develop a duplex real-time quantitative PCR assay for simultaneous detection of West Nile virus and Chikungunya virus.Methods Two sets of primers and TaqMan probes were designed based on highly conserved CAP gene region of West Nile virus and E1 gene region of Chikungunya virus,and the reactive condition of duplex real-time PCR was optimized.The sensitivity and specificity of the assay were evaluated.Results The duplex real-time quantitative PCR assay showed excellent specificity in simultaneous detection of West Nile virus and Chikungunya virus.The sensitivities of the assay were 10 copies/μL for both the virus,and the correlation coefficient of the quantitative curve were 0.999 and 0.998 respectively.Conclusion The duplex fluorescent quantitative PCR assay developed in this study is sensitive and specific for simultaneous detection of West Nile virus and Chikungunya virus.The efficiency of the assay for detecting of clinical samples should be further evaluated.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  20. Analysis of Real-time Fluorescence Quantitative PCR Detection of HBV DNA Indoor Quality Control%实时荧光定量PCR检测HBV DNA室内质控分析

    Institute of Scientific and Technical Information of China (English)

    陆小玲; 周鸣桢

    2014-01-01

    目的探讨实时荧光定量PCR在HBV DNA定量检测过程中的室内质控问题。方法用实时荧光定量PCR法检测2013年1~7月自制的乙肝阳性室内质控血清,与临床标本同步检测,计算每次阳性质控结果的常用对数、标准曲线的斜率、截距和相关系数(r)及相关结果的均值(x)、标准差(s)和变异系数(cv),利用EXCEL折线散点图画出质控图进行质控。结果本室2013年1~7月测定自制的阳性室内质控结果测定值均在控,标准差和变异系数均在允许范围内,符合要求。结论本实验室采用实时荧光定量PCR检测HBV-DNA定量检测过程中,选用的质控方法和自制的质控品稳定性良好,能可靠有效地为临床提供准确的检验结果。%Objective To explore the indoor quality controlproblems in HBV DNA quantitative detection of real-time fluorescence quantitative PCR. Indoor quality control serum HBsAg positive self detection.Methods For 1~7 months of 2013 by real-time fluorescence quantitativePCR method and clinical specimens, synchronous detection, curve of standard logarithm, calculated for eachpositive quality control results of the slope, intercept and correlation coefficient (R) value and related results ,standard deviation (s) and the coefficient of variation(CV), scat er picture quality control chart for quality control by using the EXCEL line. Results The 1~7 months of 2013 were positive results of internal quality control of homemade measured values are in control, the standard deviation and variation coefficient were within the al owable range, meet the requirements. Conclusion This laboratory detection of HBV-DNA quantitative real-time fluorescence quantitative PCR detection process,quality control methods and quality control of homemadegood stability, can provide valid and reliable accurate test results for clinical.

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

    Science.gov (United States)

    2005-10-01

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

  2. Clinical application of real-time fluorescence quantitive PCR for detecting Streptococcus pneumoniae%探针荧光定量PCR在肺炎链球菌检测中的应用

    Institute of Scientific and Technical Information of China (English)

    曹东林; 胡亮彬; 林茂锐; 王婷; 黄基伟; 田军章

    2014-01-01

    Objective To establish an assay for the detection of Streptococcus pneumoniae by real-time fluorescence quantititive polymerase chain reaction (PCR).Methods Special primers and probe for the autolysin A (lytA)gene were designed.The sensitivity and specificity of primers and probe were studied,and cut-off of cycle threshold was assayed.158 clinical specimens were confirmed by real-time fluorescence quantitative PCR and bacterial culture method.Results Primer and probe design for LytA gene could sensitively detect serotype Streptococcus pneumoniae strains of common pathogenic,and the sensitivity was 100 copies.Among 35 strains of Streptococcus pneumoniae,34 cases were detected to be positive for Streptococcus pneumoniae by real-time fluorescence quantitative PCR,while 1 case was detected to be negative;among 15 strains of non-Streptococcus pneumoniae, all were detected to be negative.Among the 158 clinical sputum specimens,34 cases with Streptococcus pneumoniae were detected by real-time fluorescence quantitative PCR,while only 10 cases with Streptococcus pneumoniae were detected by the culture method.White blood cells count and time in hospital of cases with Streptococcus pneumoniae were higher than those of cases without Streptococcus pneumoniae (P <0.05 ). Conclusion Real-time fluorescence quantitative PCR is a sensitive and specific assay for the detection of Streptococcus pneumoniae.It can be used for the diagnosis of Streptococcus pneumoniae.%目的:建立探针荧光定量PCR检测肺炎链球菌的方法。方法针对肺炎链球菌种属特异性基因lytA,设计合成了特异引物和探针,研究引物和探针的灵敏度和特异性,确定循环阈值(cycle threshold,ct)的临界值(cut-off value)。将荧光定量PCR和细菌培养法进行比较,同时检测158份肺炎患者痰液标本加以验证。结果针对LytA基因所设计的引物和探针能灵敏地检出常见致病的血清型肺炎链球菌株

  3. 实时荧光定量 PCR法分析结核分枝杆菌对异烟肼耐药的分析%Analysis of Mycobacterium tuberculosis resistant to isoniazid by real-time quantitative PCR

    Institute of Scientific and Technical Information of China (English)

    孙京涛; 孙丽娜

    2016-01-01

    目的:利用实时荧光定量PCR技术快速检测异烟肼耐药结核分枝杆菌。方法收集到医院就诊的结核病疑似患者痰液样本,提取痰液样本的总DNA,利用实时荧光定量PCR( Real-time PCR)技术对结核分枝杆菌感染进行快速筛查,并与传统药敏试验进行比较,对两者的灵敏度、特异性、一致性进行比较分析。结果检测346例结核病人临床分离培养样本,药敏试验检出257例异烟肼敏感标本,101例异烟肼耐药标本;实时荧光定量PCR 法共检测出异烟肼敏感和耐药标本225例98例,灵敏度为86.64%,特异性为93.92%,一致率为93.12%。结论跟传统药物敏感性实验相比,实时荧光定量PCR法检测速度快速、特异性强、灵敏度较高,可用于结核分枝杆菌耐异烟肼突变的快速检测,适于耐多药结核病的快速筛查。%Objective To rapid detect isoniazid resistant Mycobacterium tuberculosis by using real -time flu-orescence quantitative PCR technology .Methods The sputum samples of suspected TB patients to extract total DNA by real-time fluorescence quantitative PCR technique for rapid screening of Mycobacterium tuberculosis infection , and then it was compared with conventional drug susceptibility test in sensitivity , specificity , and consistency .Results There were 257 cases of isoniazid sensitive samples and 101 cases of isoniazid resistant samples by drug sensitivity test, and 225 cases of isoniazid sensitive samples and 98 cases of isoniazid resistant samples by real-time fluorescence quantitative PCR.The sensitivity was 86.64%, the specificity was 93.92%, and the concordance was 93.12%. Conclusion Compared with the traditional drug sensitivity test , real-time fluorescence quantitative PCR method has the advantages of quick detection and high specificity and sensitivity for Mycobacterium tuberculosis isoniazid resistant mutation detection , and it is also suitable for rapid screening

  4. Development and validation of a real-time two-step RT-qPCR TaqMan(®) assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honey bee colonies

    DEFF Research Database (Denmark)

    Blanchard, Philippe; Guillot, Sylvain; Antùnez, Karina

    2014-01-01

    Sacbrood virus (SBV) is the causal agent of a disease of honey bee larvae, resulting in failure to pupate and causing death. The typical clinical symptom of SBV is an accumulation of SBV-rich fluid in swollen sub-cuticular pouches, forming the characteristic fluid-filled sac that gives its name...... bees. A two-step real-time RT-PCR assay, based on TaqMan(®) technology using a fluorescent probe (FAM-TAMRA) was therefore developed to quantify Sacbrood virus in larvae, pupae and adult bees from symptomatic apiaries. This assay was first validated according to the recent XP-U47-600 standard issued...... with individuals without clinical signs. The SBV quantitation revealed that, in symptomatic larvae, the virus load was significantly higher than in samples without clinical signs. Combining quantitation with clinical data, a threshold of SBV viral load related to an overt disease was proposed (10(10) SBV genome...

  5. Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera

    Directory of Open Access Journals (Sweden)

    Amiteye Samuel

    2011-08-01

    Full Text Available Abstract Background Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates. Results Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18, Efalpha1 (Elongation factor 1 alpha, ACT 2 (Actin2, UBQ (polyubiquitin, PEX4 (Peroxisomal ubiquitin conjugating enzyme and At1g09770.1 (Arabidopsis thaliana cell division cycle 5. Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEfα1 or BoechUBQ and BoechEfα1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed

  6. Doubling Throughput of a Real-Time PCR

    OpenAIRE

    Christian D. Ahrberg; Pavel Neužil

    2015-01-01

    The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one...

  7. Detection of Expression Intensity of Aspergillus flavus Resistance Gene in Peanut Seed Capsule by Quantitative Real -Time PCR%荧光量PCR技术检测花生种皮抗黄曲霉基因表达强度

    Institute of Scientific and Technical Information of China (English)

    张廷婷; 李春娟; 闫彩霞; 孙兵; 郑奕雄; 单世华

    2012-01-01

    依据已获得的基因芯片和cDNA研究结果,筛选上调表达并且表达差异系数在5.0以上的花生种皮基因序列设计引物,采用SYBR Green Ⅰ实时荧光定量PCR法对上述基因进行相对定量分析,以验证基因芯片研究结果并为分离黄曲霉抗性相关基因奠定基础.结果表明:荧光定量PCR的结果与基因芯片的结果高度一致,目的基因在感病和抗病品种之间差异显著,其中BE87、AW03在抗病品种中相对表达量较高,可能在抵御黄曲霉侵染过程中发挥重要作用.%According to the research results of obtained gene chip and cDNA library, the primers were designed based on the gene sequences of peanut seed capsule which up - regulated expression and the differential expression coefficient more than 5.0. The relative quantitative analysis of these genes was carried out by SYBR Green I quantitative real - time PCR to identify the research results of gene chip and provide foundation for separating related genes resistant to Aspergillus flavus. The results showed that the quantitative real -time PCR results were consistent with the research results of gene chip on a high degree; there were significant differences between Aspergillus flavus resistant and susceptible varieties. Among which, BES1 and j4WD3 expressed more in Aspergillus flavus resistant varieties and maybe play an important role in defense from Aspergillus flavus infection.

  8. A novel strategy to obtain quantitative data for modelling: Combined enrichment and real-time PCR for enumeration of salmonellae from pig carcasses

    DEFF Research Database (Denmark)

    Malorny, Burkhard; Krämer, Nadine; Vigre, Håkan

    2011-01-01

    The primary sources for the major zoonotic pathogen Salmonella are food-producing animals such as pigs and poultry. For risk assessment and hazard analysis and critical control point (HACCP) concepts, it is essential to produce large amounts of quantitative data, which is currently not achievable...

  9. Evaluation and Selection of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis of Gene Expression in Nile Tilapia (Oreochromis niloticus) during Vaccination and Infection.

    Science.gov (United States)

    Wang, Erlong; Wang, Kaiyu; Chen, Defang; Wang, Jun; He, Yang; Long, Bo; Yang, Lei; Yang, Qian; Geng, Yi; Huang, Xiaoli; Ouyang, Ping; Lai, Weimin

    2015-04-30

    qPCR as a powerful and attractive methodology has been widely applied to aquaculture researches for gene expression analyses. However, the suitable reference selection is critical for normalizing target genes expression in qPCR. In the present study, six commonly used endogenous controls were selected as candidate reference genes to evaluate and analyze their expression levels, stabilities and normalization to immune-related gene IgM expression during vaccination and infection in spleen of tilapia with RefFinder and GeNorm programs. The results showed that all of these candidate reference genes exhibited transcriptional variations to some extent at different periods. Among them, EF1A was the most stable reference with RefFinder, followed by 18S rRNA, ACTB, UBCE, TUBA and GAPDH respectively and the optimal number of reference genes for IgM normalization under different experiment sets was two with GeNorm. Meanwhile, combination the Cq (quantification cycle) value and the recommended comprehensive ranking of reference genes, EF1A and ACTB, the two optimal reference genes, were used together as reference genes for accurate analysis of immune-related gene expression during vaccination and infection in Nile tilapia with qPCR. Moreover, the highest IgM expression level was at two weeks post-vaccination when normalized to EF1A, 18S rRNA, ACTB, and EF1A together with ACTB compared to one week post-vaccination before normalizing, which was also consistent with the IgM antibody titers detection by ELISA.

  10. PCR-反向点杂交基因分型与实时荧光定量PCR检测人乳头瘤病毒的研究%Use of a PCR-based reverse blot hybridization assay for subtyping and real-time quantitative PCR to detect human papilloma virus

    Institute of Scientific and Technical Information of China (English)

    向华国; 曾锦婷; 何婉意; 黎国

    2012-01-01

    Objective To evaluate the significance of a PCR-based reverse blot hybridization (PCR-RDB) assay and realtime quantitative PCR for detecting human papilloma virus in female outpatients. Methods A total of 121 female outpatients were checked for 23 HFV DNA types by PCR-RDB and 13 high-risk HPV genotypes by real-time quantitative PCR. Results According to PCR-RDB, 28.10% of the women(34/121) tested positive while 16. 53%(20/121) tested positive according to real-time quantitative PCR. HPV was detected more often with PCR-RDB than with real-time quantitative PCR (P<0.05). The concordance rate for the two techniques was 93. 39%(113/121). Conclusion PCR-RDB can be used to screen for HPV infection while real-time quantitative PCR facilitates evaluation of the effectiveness of treatment and the prognosis for cervical carcinoma. Combining the two should increase the specificity and sensitivity of HPV detection.%目的 评价PCR-反向点杂交基因分型与实时荧光定量PCR在检测人乳头瘤病毒(HPV)的意义.方法 同时采用PCR-反向点杂交基因分型和实时荧光定量PCR对121例女性官颈脱离细胞标本进行HPV检测.其中PCR-反向点杂交基因分型能检测23种HPV亚型,实时荧光定量PCR定量检测常见的13种高危HPV亚型.结果 PCR-反向点杂交基因分型检测HPV的阳性率为28.10%(34/121),实时荧光定量PCR检测HPV的阳性率为16.53%(20/121),差异有统计学意义(P<0.05);二者检测的符合率为93.39%(113/121).结论 PCR-反向杂交基因分型适用于HPV感染的筛查,而实时荧光定量PCR适用于HPV感染相关疾病的疗效与预后的判断.PCR-反向杂交基因分型与实时荧光定量PCR联合检测可提高HPV检测的特异性和敏感度,对于生殖道HPV感染以及子宫颈癌的早期发现、预防和治疗具有重要意义.

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

    Science.gov (United States)

    Southard, Jonathan N.

    2014-01-01

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

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

    Science.gov (United States)

    Southard, Jonathan N.

    2014-01-01

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

  13. PCR real time assays for the early detection of BKV-DNA in immunocompromised patients.

    Science.gov (United States)

    Marinelli, Katia; Bagnarelli, Patrizia; Gaffi, Gianni; Trappolini, Silvia; Leoni, Pietro; Paggi, Alessandra Mataloni; Della Vittoria, Agnese; Scalise, Giorgio; Varaldo, Pietro Emanuele; Menzo, Stefano

    2007-07-01

    Testing for viral BKV-DNA in urine is a non-invasive early detection and monitoring tool in the diagnostic of BKV-related pathologies: quantitative analysis by Real-Time PCR can provide useful information in addition to cytologic analysis, although our study suggests that high BKV viruria is not necessarily associated with kidney or bladder damage.

  14. Electrochemistry-based real-time PCR on a microchip.

    Science.gov (United States)

    Yeung, Stephen S W; Lee, Thomas M H; Hsing, I-Ming

    2008-01-15

    The development of handheld instruments for point-of-care DNA analysis can potentially contribute to the medical diagnostics and environmental monitoring for decentralized applications. In this work, we demonstrate the implementation of a recently developed electrochemical real-time polymerase chain reaction (ERT-PCR) technique on a silicon-glass microchip for simultaneous DNA amplification and detection. This on-chip ERT-PCR process requires the extension of an oligonucleotide in both solution and at solid phases and intermittent electrochemical signal measurement in the presence of all the PCR reagents. Several important parameters, related to the surface passivation and electrochemical scanning of working electrodes, were investigated. It was found that the ERT-PCR's onset thermal cycle ( approximately 3-5), where the analytical signal begins to be distinguishable from the background, is much lower than that of the fluorescence-based counterparts for high template DNA situations (3 x 10(6) copies/microL). By carefully controlling the concentrations of the immobilized probe and the enzyme polymerase, improvements have been made in obtaining a meaningful electrochemical signal using a lower initial template concentration. This ERT-PCR technique on a microchip platform holds significant promise for rapid DNA detection for point-of-care testing applications.

  15. Cytokine mRNA quantification by real-time PCR.

    Science.gov (United States)

    Stordeur, Patrick; Poulin, Lionel F; Craciun, Ligia; Zhou, Ling; Schandené, Liliane; de Lavareille, Aurore; Goriely, Stanislas; Goldman, Michel

    2002-01-01

    Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.

  16. Multiplex real-time PCR assay for Legionella species.

    Science.gov (United States)

    Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja

    2015-12-01

    Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies.

  17. Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR

    Science.gov (United States)

    Eissa, Nour; Kermarrec, Laëtitia; Hussein, Hayam; Bernstein, Charles N.; Ghia, Jean-Eric

    2017-01-01

    2,4-Dinitrobenzene sulfonic acid (DNBS)-induced colitis is an experimental model that mimics Crohn’s disease. Appropriateness of reference genes is crucial for RT-qPCR. This is the first study to determine the stability of reference gene expression (RGE) in mice treated with DNBS. DNBS experimental Colitis was induced in male C57BL/6 mice. RNA was extracted from colon tissue and comprehensive analysis of 13 RGE was performed according to predefined criteria. Relative colonic TNF-α and IL-1β mRNA levels were calculated. Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest fluctuation within the inflamed and control groups. Conversely, ribosomal protein large P0 (Rplp0), non-POU domain containing (Nono), TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. TNF-α and IL-1β mRNA levels was dependent on the reference gene used and varied from significant when the most stable genes were used to non-significant when the least stable genes were used. The appropriate choice of RGE is critical to guarantee satisfactory normalization of RT-qPCR data when using DNBS-Model. We recommend using Rplp0, Nono, Tbp, Hprt and Eef2 instead of common reference genes. PMID:28186172

  18. Selection of reference genes for expression analysis using quantitative real-time PCR in the pea aphid, Acyrthosiphon pisum (Harris (Hemiptera, Aphidiae.

    Directory of Open Access Journals (Sweden)

    Chunxiao Yang

    Full Text Available To facilitate gene expression study and obtain accurate qRT-PCR analysis, normalization relative to stable expressed housekeeping genes is required. In this study, expression profiles of 11 candidate reference genes, including actin (Actin, elongation factor 1 α (EF1A, TATA-box-binding protein (TATA, ribosomal protein L12 (RPL12, β-tubulin (Tubulin, NADH dehydrogenase (NADH, vacuolar-type H+-ATPase (v-ATPase, succinate dehydrogenase B (SDHB, 28S ribosomal RNA (28S, 16S ribosomal RNA (16S, and 18S ribosomal RNA (18S from the pea aphid Acyrthosiphon pisum, under different developmental stages and temperature conditions, were investigated. A total of four analytical tools, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the suitability of these genes as endogenous controls. According to RefFinder, a web-based software tool which integrates all four above-mentioned algorithms to compare and rank the reference genes, SDHB, 16S, and NADH were the three most stable house-keeping genes under different developmental stages and temperatures. This work is intended to establish a standardized qRT-PCR protocol in pea aphid and serves as a starting point for the genomics and functional genomics research in this emerging insect model.

  19. Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis

    DEFF Research Database (Denmark)

    Andersen, Ulla Vestergaard; Haakansson, I. T.; Roust, Tina

    2013-01-01

    strongyle type parasites (NC1, NC2). SYBR Green Real-Time PCRs were performed in triplicates. Results revealed a statistically significant increase in PCR yield after three days, which was statistically associated with beginning embryonation of the eggs. In conclusion, storage time and developmental stage...... of strongyle eggs are significant sources of error in studies based on quantitative real-time PCR analysis. This study suggests that for refrigerated storage of more than three days, eggs should be inactivated and preserved for further analysis....

  20. Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires' disease.

    Science.gov (United States)

    Mentasti, M; Fry, N K; Afshar, B; Palepou-Foxley, C; Naik, F C; Harrison, T G

    2012-08-01

    The detection of Legionella pneumophila DNA in clinical specimens using quantitative real-time polymerase chain reaction (qPCR) combined with direct sequence-based typing (SBT) offers rapid confirmation and timely intervention in the investigation of cases of Legionnaires' disease (LD). We assessed the utility of a specific L. pneumophila qPCR assay targeting the macrophage infectivity potentiator (mip) gene and internal process control with three clinical specimen types from confirmed LD cases. The assay was completely specific for L. pneumophila, as demonstrated by positive results for 39/39 strains from all subspecies and 16 serogroups. No cross-reaction was observed with any of the 54 Legionella non-pneumophila (0/69 strains) or 21 non-Legionella (0/58 strains). All L. pneumophila culture-positive respiratory samples (81/81) were qPCR-positive. Of 80 culture-negative samples tested, 47 (58.8%) were qPCR-positive and none were inhibitory. PCR was significantly more sensitive than culture for samples taken ≤ 2 days of hospitalisation (94.7% vs. 79.6%), with the difference being even more marked for samples taken between 3 and 14 days (79.3% vs. 47.8%). Overall, the sensitivity of the qPCR was ∼30% greater than that of culture and direct typing on culture-negative PCR-positive samples resulted in full 7-allele profiles from 23/46, 5 to 6 alleles from 8/46 and ≥ 1 allele from 43/46 strains.

  1. Real-Time PCR using a PCR Microchip with Integrated Thermal System and Polymer Waveguides for the Detection of Campylobacter jejuni

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Sekulovic, Andrea; Kutter, Jörg Peter

    2006-01-01

    A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. By using the integrated optical system of the real-time PCR chip, cadF – a virulence gene of Campylobacter jejuni, could specifically be detected. Two different DNA binding dyes, SYTOX...... Orange and TO-PRO-3, were added to the PCR mixture to realize the real-time PCR. The presented approach shows reliable real-time quantitative information of the PCR amplification of the targeted gene....

  2. Evaluation of sorghum [Sorghum bicolor (L.] reference genes in various tissues and under abiotic stress conditions for quantitative real-time PCR data normalization