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Sample records for analysis quantitative pcr

  1. A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis.

    PubMed

    Funes-Huacca, Maribel E; Opel, Kerry; Thompson, Robyn; McCord, Bruce R

    2011-04-01

    In this paper we compare the effects of three representative PCR inhibitors using quantitative PCR (qPCR) and multiplex STR amplification in order to determine the effect of inhibitor concentration on allele dropout and to develop better ways to interpret forensic DNA data. We have used humic acid, collagen and calcium phosphate at different concentrations to evaluate the profiles of alleles inhibited in these amplifications. These data were correlated with previously obtained results from quantitative PCR including melt curve effects, efficiency changes and cycle threshold (Ct) values. Overall, the data show that there are two competing processes that result from PCR inhibition. The first process is a general loss of larger alleles. This appears to occur with all inhibitors. The second process is more sequence specific and occurs when the inhibitor binds DNA, altering the cycle threshold and the melt curve. This sequence-specific inhibition results in patterns of allele loss that occur in addition to the overall loss of larger alleles. The data demonstrate the applicability of utilizing real-time PCR results to predict the presence of certain types of PCR inhibition in STR analysis. PMID:21462225

  2. Quantitative PCR analysis of laryngeal muscle fiber types

    PubMed Central

    Van Daele, Douglas J.

    2013-01-01

    Voice and swallowing dysfunction as a result of recurrent laryngeal nerve paralysis can be improved with vocal fold injections or laryngeal framework surgery. However, denervation atrophy can cause late-term clinical failure. A major determinant of skeletal muscle physiology is myosin heavy chain (MyHC) expression, and previous protein analyses have shown changes in laryngeal muscle fiber MyHC isoform with denervation. RNA analyses in this setting have not been performed, and understanding RNA levels will allow interventions better designed to reverse processes such as denervation in the future. Total RNA was extracted from bilateral rat thyroarytenoid (TA), posterior cricoarytenoid (PCA), and cricothyroid (CT) muscles in rats. Primers were designed using published MyHC isoform sequences. SYBR Green real time reverse transcription-polymerase chain reaction (SYBR-RT-PCR) was used for quantification. The electropherogram showed a clear separation of total RNA to 28S and 18S subunits. Melting curves illustrated single peaks for all type MyHC primers. All MyHC isoforms were identified in all muscles with various degrees of expression. Quantitative PCR is a sensitive method to detect MyHC isoforms in laryngeal muscle. Isoform expression using mRNA analysis was similar to previous analyses but showed some important differences. This technique can be used to quantitatively assess response to interventions targeted to maintain muscle bulk after denervation. PMID:20430402

  3. MOLD SPECIFIC QUANTITATIVE PCR: THE EMERGING STANDARD IN MOLD ANALYSIS

    EPA Science Inventory

    Today I will talk about the use of quantitative or Real time PCR for the standardized identification and quantification of molds. There are probably at least 100,000 species of molds or fungi. But there are actually about 100 typically found indoors. Some pose a threat to human...

  4. Quantitative PCR analysis of salivary pathogen burden in periodontitis

    PubMed Central

    Salminen, Aino; Kopra, K. A. Elisa; Hyvärinen, Kati; Paju, Susanna; Mäntylä, Päivi; Buhlin, Kåre; Nieminen, Markku S.; Sinisalo, Juha; Pussinen, Pirkko J.

    2015-01-01

    Our aim was to investigate the value of salivary concentrations of four major periodontal pathogens and their combination in diagnostics of periodontitis. The Parogene study included 462 dentate subjects (mean age 62.9 ± 9.2 years) with coronary artery disease (CAD) diagnosis who underwent an extensive clinical and radiographic oral examination. Salivary levels of four major periodontal bacteria were measured by quantitative real-time PCR (qPCR). Median salivary concentrations of Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, as well as the sum of the concentrations of the four bacteria, were higher in subjects with moderate to severe periodontitis compared to subjects with no to mild periodontitis. Median salivary Aggregatibacter actinomycetemcomitans concentrations did not differ significantly between the subjects with no to mild periodontitis and subjects with moderate to severe periodontitis. In logistic regression analysis adjusted for age, gender, diabetes, and the number of teeth and implants, high salivary concentrations of P. gingivalis, T. forsythia, and P. intermedia were significantly associated with moderate to severe periodontitis. When looking at different clinical and radiographic parameters of periodontitis, high concentrations of P. gingivalis and T. forsythia were significantly associated with the number of 4–5 mm periodontal pockets, ≥6 mm pockets, and alveolar bone loss (ABL). High level of T. forsythia was associated also with bleeding on probing (BOP). The combination of the four bacteria, i.e., the bacterial burden index, was associated with moderate to severe periodontitis with an odds ratio (OR) of 2.40 (95% CI 1.39–4.13). When A. actinomycetemcomitans was excluded from the combination of the bacteria, the OR was improved to 2.61 (95% CI 1.51–4.52). The highest OR 3.59 (95% CI 1.94–6.63) was achieved when P. intermedia was further excluded from the combination and only the levels of P. gingivalis and

  5. Analysis of copy number variation using quantitative interspecies competitive PCR.

    PubMed

    Williams, Nigel M; Williams, Hywel; Majounie, Elisa; Norton, Nadine; Glaser, Beate; Morris, Huw R; Owen, Michael J; O'Donovan, Michael C

    2008-10-01

    Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls. PMID:18697816

  6. Quantitative analysis of food and feed samples with droplet digital PCR.

    PubMed

    Morisset, Dany; Štebih, Dejan; Milavec, Mojca; Gruden, Kristina; Žel, Jana

    2013-01-01

    In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed. PMID:23658750

  7. Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR

    PubMed Central

    Morisset, Dany; Štebih, Dejan; Milavec, Mojca; Gruden, Kristina; Žel, Jana

    2013-01-01

    In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed. PMID:23658750

  8. QUANTITATIVE PCR ANALYSIS OF MOLDS IN THE DUST FROM HOMES OF ASTHMATIC CHILDREN IN NORTH CAROLINA

    EPA Science Inventory

    The vacuum bag (VB) dust was analyzed by mold specific quantitative PCR. These results were compared to the analysis survey calculated for each of the homes. The mean and standard deviation (SD) of the ERMI values in the homes of the NC asthmatic children was 16.4 (6.77), compa...

  9. Quantitative Analysis of Pork and Chicken Products by Droplet Digital PCR

    PubMed Central

    Cai, Yicun; Li, Xiang; Lv, Rong; Yang, Jielin; Li, Jian; He, Yuping; Pan, Liangwen

    2014-01-01

    In this project, a highly precise quantitative method based on the digital polymerase chain reaction (dPCR) technique was developed to determine the weight of pork and chicken in meat products. Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of species-specific DNAs in meat products. However, it is limited in amplification efficiency and relies on standard curves based Ct values, detecting and quantifying low copy number target DNA, as in some complex mixture meat products. By using the dPCR method, we find the relationships between the raw meat weight and DNA weight and between the DNA weight and DNA copy number were both close to linear. This enabled us to establish formulae to calculate the raw meat weight based on the DNA copy number. The accuracy and applicability of this method were tested and verified using samples of pork and chicken powder mixed in known proportions. Quantitative analysis indicated that dPCR is highly precise in quantifying pork and chicken in meat products and therefore has the potential to be used in routine analysis by government regulators and quality control departments of commercial food and feed enterprises. PMID:25243184

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

    PubMed Central

    Shain, Eric B.; Clemens, John M.

    2008-01-01

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

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

    PubMed

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

    2009-06-01

    A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this. PMID:19602858

  12. Development of qualitative and quantitative PCR analysis for meat adulteration from RNA samples.

    PubMed

    Cheng, Jai-Hong; Chou, Hsiao-Ting; Lee, Meng-Shiou; Sheu, Shyang-Chwen

    2016-02-01

    Total RNA samples were used to establish qualitative and quantitative PCR-based methods for assessing meat adulteration. The primers were designed based on the mRNA sequences of troponin I (TnI), mitochondrial ribosomal protein (MRP) and tropomodulin genes to distinguish chicken, pork, goat, beef and ostrich. There was no cross reaction between the primers, and the detection limit of the cDNA template was 0.01 and 20 ng in simplex PCR and multiplex PCR, respectively. In the low temperature storage test, the detection limits of cDNA template with 10 and 1 ng were determined at 4 °C and -80 °C. In quantitative assay, the precision of real-time PCR analysis expressed as a coefficient of variation (CV) ranged from 0.25% to 5.24% and the trueness, expressed as an error, ranged from 0.28% to 6.98% for adulteration. Thus, herein, we provided alternative tools for the assessment of meat adulteration using mRNA-based PCR methods. PMID:26304356

  13. Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution.

    PubMed

    Weaver, Suzanne; Dube, Simant; Mir, Alain; Qin, Jian; Sun, Gang; Ramakrishnan, Ramesh; Jones, Robert C; Livak, Kenneth J

    2010-04-01

    This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis. PMID:20079846

  14. Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater

    EPA Science Inventory

    A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. Thes...

  15. Quantitative Analysis of Copy Number Variants Based on Real-Time LightCycler PCR

    PubMed Central

    Ma, Lijiang; Chung, Wendy K.

    2014-01-01

    Quantitative real-time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes used to measure gene expression or gene quantification including including contiguous gene deletions or duplications. A simple method is described to quantify DNA copy number from human samples. PMID:24510682

  16. Comparative analysis of techniques for detection of quiescent Botrytis cinerea in grapes by quantitative PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative PCR (qPCR) can be used to detect and monitor pathogen colonization, but early attempts to apply the technology to quiescent Botrytis cinerea infections of grape berries identified some specific limitations. In this study, four DNA extraction methods, two tissue-grinding methods, two gra...

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of roundup ready soybean.

    PubMed

    Zhang, Haibo; Yang, Litao; Guo, Jinchao; Li, Xiang; Jiang, Lingxi; Zhang, Dabing

    2008-07-23

    To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs. However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis. In this study, we developed one unique plasmid molecule based on one pMD-18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), that is, CaMV35S, NOS, and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then, we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting CaMV35S, NOS, and RRS events employing this reference molecule as the calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 and 50 copies, respectively. For the quantitative analysis of practical RRS samples, the results of accuracy and precision were similar to those of simplex PCR assays, for instance, the quantitative results were at the 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6%, respectively, and the statistic analysis ( t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs. PMID:18570432

  20. Quantitative PCR analysis of CYP1A induction in Atlantic salmon (Salmo salar)

    USGS Publications Warehouse

    Rees, C.B.; McCormick, S.D.; Vanden, Heuvel J.P.; Li, W.

    2003-01-01

    Environmental pollutants are hypothesized to be one of the causes of recent declines in wild populations of Atlantic salmon (Salmo salar) across Eastern Canada and the United States. Some of these pollutants, such as polychlorinated biphenyls and dioxins, are known to induce expression of the CYP1A subfamily of genes. We applied a highly sensitive technique, quantitative reverse transcription-polymerase chain reaction (RT-PCR), for measuring the levels of CYP1A induction in Atlantic salmon. This assay was used to detect patterns of CYP1A mRNA levels, a direct measure of CYP1A expression, in Atlantic salmon exposed to pollutants under both laboratory and field conditions. Two groups of salmon were acclimated to 11 and 17??C, respectively. Each subject then received an intraperitoneal injection (50 mg kg-1) of either ??-naphthoflavone (BNF) in corn oil (10 mg BNF ml-1 corn oil) or corn oil alone. After 48 h, salmon gill, kidney, liver, and brain were collected for RNA isolation and analysis. All tissues showed induction of CYP1A by BNF. The highest base level of CYP1A expression (2.56??1010 molecules/??g RNA) was found in gill tissue. Kidney had the highest mean induction at five orders of magnitude while gill tissue showed the lowest mean induction at two orders of magnitude. The quantitative RT-PCR was also applied to salmon sampled from two streams in Massachusetts, USA. Salmon liver and gill tissue sampled from Millers River (South Royalston, Worcester County), known to contain polychlorinated biphenyls (PCBs), showed on average a two orders of magnitude induction over those collected from a stream with no known contamination (Fourmile Brook, Northfield, Franklin County). Overall, the data show CYP1A exists and is inducible in Atlantic salmon gill, brain, kidney, and liver tissue. In addition, the results obtained demonstrate that quantitative PCR analysis of CYP1A expression is useful in studying ecotoxicity in populations of Atlantic salmon in the wild. ?? 2003

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

    PubMed

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

    2003-01-01

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

  2. QUANTITATIVE PCR ANALYSIS OF HOUSE DUST CAN REVEAL ABNORMAL MOLD CONDITIONS

    EPA Science Inventory

    Indoor mold populations were measured in the dust of homes in Cleveland and Cincinnati, OH, by quantitative PCR (QPCR) and, in Cincinnati, also by culturing. QPCR assays for 82 species (or groups of species) were used to identify and quantify indoor mold populations in moldy home...

  3. Quantitative PCR Analysis of Molds in the Dust from Homes of Asthmatic Children in North Carolina

    SciTech Connect

    Vesper, Stephen J.; McKinstry, Craig A.; Ashley, Peter; Haugland, Richard A.; Yeatts, Karin; Bradham, Karen; Svendsen, Eric

    2007-07-10

    The vacuum cleaner bag (VCB) dust from the homes of 19 asthmatic children in North Carolina (NC) was analyzed by mold specific quantitative PCR. These results were compared to the analysis of the VCB dust from 157 homes in the HUD “American Healthy Home Survey” of homes in the US. The American Relative Moldiness Index (ARMI) was calculated for each of the homes. The mean and standard deviation (SD) of the ARMI values in the homes of the NC asthmatic children was 11.0 (5.3), compared to the HUD survey VCB ARMI value mean and SD of 6.6 (4.4). The median ARMI value was significantly higher(p < 0.001) in the asthmatic childrens’s homes. The molds Chaetomium globosum and Eurotium amsterdameli were the primary species in the NC homes making the ARMI values higher. Vacuum cleaner bag dust samples may be a less expensive but still useful method of home mold analysis.

  4. EVALUATION OF RAPID DNA EXTRACTION PROCEDURES FOR THE QUANTITATIVE DETECTION OF FUNGAL CELLS USING REAL TIME PCR ANALYSIS

    EPA Science Inventory

    The ease and rapidity of quantitative DNA sequence detection by real-time PCR instruments promises to make their use increasingly common for the microbial analysis many different types of environmental samples. To fully exploit the capabilities of these instruments, correspondin...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2014-01-01

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

  7. Quantitative PCR analysis of house dust can reveal abnormal mold conditions†

    PubMed Central

    Meklin, Teija; Haugland, Richard A.; Reponen, Tiina; Varma, Manju; Lummus, Zana; Bernstein, David; Wymer, Larry J.; Vesper, Stephen J.

    2007-01-01

    Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were > 1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR. PMID:15237292

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

    PubMed Central

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

    2015-01-01

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

  9. Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae).

    PubMed

    Piron Prunier, Florence; Chouteau, Mathieu; Whibley, Annabel; Joron, Mathieu; Llaurens, Violaine

    2016-01-01

    Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms. PMID:27271971

  10. Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)

    PubMed Central

    Chouteau, Mathieu; Whibley, Annabel; Joron, Mathieu; Llaurens, Violaine

    2016-01-01

    Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata. This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms. PMID:27271971

  11. Quantitative Real-Time PCR Analysis of Gene Transcripts of Mosquito Follicles.

    PubMed

    Telang, Aparna

    2016-01-01

    Real-time (quantitative) PCR, or QPCR, has become an indispensible tool for characterizing gene expression. Depending on the experimental design, researchers can use either the relative or absolute (standard curve) method to quantify transcript abundance. Characterizing the expression of genes in mosquito ovaries will require use of the standard curve method of quantification. Here, I describe reagents and equipment necessary to run standard curve QPCR. I also provide details on the construction of the standard linear curve and calculations required to determine transcript abundance. PMID:27557577

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

    PubMed Central

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

    2015-01-01

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

  13. A quantitative reverse transcription-PCR assay for rapid, automated analysis of breast cancer sentinel lymph nodes.

    PubMed

    Hughes, Steven J; Xi, Liqiang; Gooding, William E; Cole, David J; Mitas, Michael; Metcalf, John; Bhargava, Rohit; Dabbs, David; Ching, Jesus; Kozma, Lynn; McMillan, William; Godfrey, Tony E

    2009-11-01

    We have previously reported that a quantitative reverse transcription (QRT)-PCR assay accurately analyzes sentinel lymph nodes (SLNs) from breast cancer patients. The aim of this study was to assess a completely automated, cartridge-based version of the assay for accuracy, predictive value, and reproducibility. The triplex (two markers + control) QRT-PCR assay was incorporated into a single-use cartridge for point-of-care use on the GeneXpert system. Three academic centers participated equally. Twenty-nine positive lymph nodes and 30 negative lymph nodes were analyzed to establish classification rules. SLNs from 120 patients were subsequently analyzed by QRT-PCR and histology (including immunohistochemistry), and the predetermined decision rules were used to classify the SLNs; 112 SLN specimens produced an informative result by both QRT-PCR and histology. By histological analysis, 21 SLNs were positive and 91 SLNs were negative for metastasis. QRT-PCR characterization produced a classification with 100% sensitivity, 97.8% specificity, and 98.2% accuracy compared with histology (91.3% positive predictive value and 100% negative predictive value). Interlaboratory reproducibility analyses demonstrated that a 95% prediction interval for a new measurement (DeltaCt) ranged between 0.403 and 0.956. This fully automated QRT-PCR assay accurately characterizes breast cancer SLNs for the presence of metastasis. Furthermore, the assay is not dependent on subjective interpretation, is reproducible across three clinical environments, and is rapid enough to allow intraoperative decision making. PMID:19797614

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

    PubMed

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

    2010-01-01

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

  15. Differentiation of five body fluids from forensic samples by expression analysis of four microRNAs using quantitative PCR.

    PubMed

    Sauer, Eva; Reinke, Ann-Kathrin; Courts, Cornelius

    2016-05-01

    Applying molecular genetic approaches for the identification of forensically relevant body fluids, which often yield crucial information for the reconstruction of a potential crime, is a current topic of forensic research. Due to their body fluid specific expression patterns and stability against degradation, microRNAs (miRNA) emerged as a promising molecular species, with a range of candidate markers published. The analysis of miRNA via quantitative Real-Time PCR, however, should be based on a relevant strategy of normalization of non-biological variances to deliver reliable and biologically meaningful results. The herein presented work is the as yet most comprehensive study of forensic body fluid identification via miRNA expression analysis based on a thoroughly validated qPCR procedure and unbiased statistical decision making to identify single source samples. PMID:26878708

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

    PubMed Central

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

    2009-01-01

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

  17. PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

    EPA Science Inventory

    ABSTRACT

    Palatal Dysmorphogenesis : Quantitative RT-PCR

    Gary A. Held and Barbara D. Abbott

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

  18. Analysis of Enterococci and Bacteriodales Fecal Indicator Bacteria in a Lake Michigan Tributary by Real-Time Quantitative PCR

    EPA Science Inventory

    The Salt Creek watershed in northwest Indiana drains into Lake Michigan near several heavily used recreational beaches. This study aimed to investigate the levels of fecal indicator bacteria, enterococci and Bacteroidales, in Salt Creek using real-time quantitative PCR (qPCR) an...

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

    PubMed

    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. PMID:21873818

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

    PubMed

    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. PMID:26818982

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

    PubMed

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

    2016-01-01

    Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions. PMID:27525844

  2. Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR

    PubMed Central

    Xiao, Xinlong; Ma, Jinbiao; Wang, Junru; Wu, Xiaomeng; Li, Pengbo; Yao, Yinan

    2015-01-01

    Real-time quantitative polymerase chain reaction (RT-qPCR), a reliable technique for quantifying gene expression, requires stable reference genes to normalize its data. Salicornia europaea, a stem succulent halophyte with remarkable salt resistance and high capacity for ion accumulation, has not been investigated with regards to the selection of appropriate reference genes for RT-qPCR. In this study, the expression of 11 candidate reference genes, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), Actin, α-Tub (α-tubulin), β-Tub (β-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), UBQ (Polyubiquitin), CYP (Cyclophilin), TIP41 (TIP41-like protein), CAC (Clathrin adaptor complexes), and DNAJ (DnaJ-like protein), was analyzed in S. europaea samples, which were classified into groups according to various abiotic stresses (NaCl, nitrogen, drought, cold and heat), tissues and ages. Three commonly used software programs (geNorm, NormFinder and BestKeeper) were applied to evaluate the stability of gene expression, and comprehensive ranks of stability were generated by aggregate analysis. The results show that the relatively stable genes for each group are the following: (1) CAC and UBC for whole samples; (2) CAC and UBC for NaCl stress; (3) Actin and α-Tub for nitrogen treatment; (4) Actin and GAPDH for drought stress; (5) α-Tub and UBC for cold stress; (6) TIP41 and DNAJ for heat stress; (7) UBC and UBQ for different tissues; and (8) UBC and Actin for various developmental stages. These genes were validated by comparing transcriptome profiles. Using two stable reference genes was recommended in the normalization of RT-qPCR data. This study identifies optimal reference genes for RT-qPCR in S. europaea, which will benefit gene expression analysis under these conditions. PMID:25653658

  3. Typing of human adenoviruses in specimens from immunosuppressed patients by PCR-fragment length analysis and real-time quantitative PCR.

    PubMed

    Ebner, Karin; Rauch, Margit; Preuner, Sandra; Lion, Thomas

    2006-08-01

    Currently, 51 human adenovirus (AdV) serotypes, which are divided into six species (A to F), are known. AdV infections are a major cause of morbidity and mortality in immunosuppressed individuals, particularly in allogeneic stem cell transplant (SCT) recipients. Any AdV species may cause life-threatening disease, but little information is available on the clinical relevance of individual serotypes. The use of serological testing for serotype identification is limited due to the impaired immune response during the posttransplant period. A new molecular approach to serotype identification is presented here that exploits variable regions within the hexon gene. All serotypes belonging to the species A, B, C, E, and F can be determined by fragment length analysis of a single PCR product. For species C, which is the most prevalent in many geographic regions, an alternative technique based on serotype-specific real-time quantitative PCR was established. Of 135 consecutive pediatric patients screened for AdV infections after allogeneic SCT, 40 tested positive. Detailed analysis revealed the presence of 10 different serotypes; serotypes 1 and 2 from species C (C01 and C02) showed the highest prevalence, accounting for 77% of the AdV-positive cases. Representatives of other species were observed less commonly: serotype A12 in 6.5%; serotype A31 in 4.5%; and B03, B16, C05, C06, D19, and F41 in 2%. The approach to rapid molecular serotype analysis presented here provides a basis for detailed studies on adenovirus epidemiology and on the transmission of nosocomial infections. Moreover, in view of the increasing importance of tailored therapy approaches, serotype identification may in the future have implications for the selection of the most appropriate antiviral treatment. PMID:16891496

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2012-05-01

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

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

    PubMed Central

    2010-01-01

    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. PMID:20149225

  7. Quantitative high-resolution melting PCR analysis for monitoring of fermentation microbiota in sourdough.

    PubMed

    Lin, Xiaoxi B; Gänzle, Michael G

    2014-09-01

    Current methods of monitoring the microbial ecology of food fermentation system are generally labor intensive and/or time consuming. This study developed two methods based on high-resolution melting curves (HRM) to monitor sourdough microbiota during fermentation and to investigate the effect of cereal substrate on microbial ecology. A strain cocktail of Lactobacillus fermentum FUA3165, Lactobacillus plantarum FUA3309, Lactobacillus paracasei FUA3166 and Lactobacillus reuteri FUA3168 was used to ferment red (Town and PAN8609) and white (commercial and Segaolane) sorghum sourdough, and wheat sourdough. The microbial composition of sourdoughs was determined by plate count and HRM-qPCR to differentiate at the species level. The resistance of each species to sorghum phenolic extract was measured. There was no difference in microbial composition among the four sorghum sourdoughs, with L. fermentum FUA3165 in all sourdoughs. The competiveness of the strains in sorghum sourdoughs corresponded to their resistance to sorghum phenolic extract. In a second experiment, five L. reuteri strains, the human-lineage strains FUA3400 and 3401 isolated from wheat sourdough, the rodent-lineage strain FUA5448 isolated from rye sourdough and the sorghum isolates FUA3168 and 3324, were used to ferment wheat, rye and sorghum sourdoughs. The microbial composition of sourdoughs was determined by plate counts and HRM-qPCR to different L. reuteri strains representing different host-adapted lineages. No difference among different substrates was observed; indicating cereal type had no selective effect on sourdough microbial ecology. In conclusion, HRM-qPCR assays were established as rapid and highly specific tool for monitoring of sourdough microbiota. The ability to distinguish highly similar microbes in samples containing only few genotypes makes HRM-qPCR suitable for quality control in other food fermentation systems. The presence of phenolic compounds in sorghum sourdough favored organisms

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

    PubMed Central

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

    2015-01-01

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

  9. Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.

    PubMed

    Stefano, Biricolti; Patrizia, Bogani; Matteo, Cerboneschi; Massimo, Gori

    2016-06-01

    One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct. PMID:26895172

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

    PubMed

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

    2005-04-01

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

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

    PubMed Central

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

    2009-01-01

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

  12. Recommended Reference Genes for Quantitative PCR Analysis in Soybean Have Variable Stabilities during Diverse Biotic Stresses

    PubMed Central

    Bansal, Raman; Mittapelly, Priyanka; Cassone, Bryan J.; Mamidala, Praveen; Redinbaugh, Margaret G.; Michel, Andy

    2015-01-01

    For real-time reverse transcription-PCR (qRT-PCR) in soybean, reference genes in different tissues, developmental stages, various cultivars, and under stress conditions have been suggested but their usefulness for research on soybean under various biotic stresses occurring in North-Central U.S. is not known. Here, we investigated the expression stabilities of ten previously recommended reference genes (ABCT, CYP, EF1A, FBOX, GPDH, RPL30, TUA4, TUB4, TUA5, and UNK2) in soybean under biotic stress from Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two‐spotted spider mite (TSSM). BPMV, PMD, SBA, and TSSM are amongst the most common pest problems on soybean in North-Central U.S. and other regions. Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Reference genes showed variability in their expression as well as stability across various stressors and the best reference genes were stress-dependent. ABCT and FBOX were found to be the most stable in soybean under both BPMV and SBA stress but these genes had only minimal to moderate stability during PMD and TSSM stress. Expression of TUA4 and CYP was found to be most stable during PMD stress; TUB4 and TUA4 were stable under TSSM stress. Under various biotic stresses on soybean analyzed, GPDH expression was found to be consistently unstable. For all biotic stressors on soybean, we obtained pairwise variation (V2/3) values less than 0.15 which suggested that combined use of the two most stable reference genes would be sufficient for normalization. Further, we demonstrated the utility of normalizing the qRT-PCR data for target genes using the most stable reference genes validated in current study. Following of the recommendations from our current study will enable an accurate and reliable normalization of qRT-PCR data in soybean under biotic stress. PMID:26244340

  13. Analysis of Fungal Flora in Indoor Dust by Ribosomal DNA Sequence Analysis, Quantitative PCR, and Culture▿ †

    PubMed Central

    Pitkäranta, M.; Meklin, T.; Hyvärinen, A.; Paulin, L.; Auvinen, P.; Nevalainen, A.; Rintala, H.

    2008-01-01

    In recent years increasing attention has been given to the potential health effects of fungal exposure in indoor environments. We used large-scale sequencing of the fungal internal transcribed spacer region (ITS) of nuclear ribosomal DNA to describe the mycoflora of two office buildings over the four seasons. DNA sequencing was complemented by cultivation, ergosterol determination, and quantitative PCR analyses. Sequences of 1,339 clones were clustered into 394 nonredundant fungal operational taxonomical units containing sequences from 18 fungal subclasses. The observed flora differed markedly from that recovered by cultivation, the major differences being the near absence of several typical indoor mold genera such as Penicillium and Aspergillus spp. and a high prevalence of basidiomycetes in clone libraries. A total of 55% of the total diversity constituted of unidentifiable ITS sequences, some of which may represent novel fungal species. Dominant species were Cladosporium cladosporioides and C. herbarum, Cryptococcus victoriae, Leptosphaerulina americana and L. chartarum, Aureobasidium pullulans, Thekopsora areolata, Phaeococcomyces nigricans, Macrophoma sp., and several Malassezia species. Seasonal differences were observed for community composition, with ascomycetous molds and basidiomycetous yeasts predominating in the winter and spring and Agaricomycetidae basidiomycetes predominating in the fall. The comparison of methods suggested that the cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal flora than any of the methods would give alone. The current restrictions of the methods are discussed. PMID:17981947

  14. Comparison of a quantitative Real-Time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes.

    PubMed

    Bharuthram, Avani; Paximadis, Maria; Picton, Anabela C P; Tiemessen, Caroline T

    2014-07-01

    The controversy surrounding the findings that copy number variation, of the CCL3 encoding genes, influences HIV-1 infection and disease progression has been in part attributed to the variable results obtained from methods used for copy number evaluation. Like CCL3, the genes encoding the CC chemokine CCL4, also a natural ligand of the CCR5 receptor, are found to occur in population-specific multiple copy number and have been shown to play a protective role against HIV-1. This study evaluated the standard method of quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR) for CCL4L gene copy number determination. The CCL4 encoding genes are CCL4, occurring in two copies per diploid genome (pdg), and the non-allelic CCL4L genes, comprised of CCL4L1 and CCL4L2, which are both found in multiple copies pdg. Copy number of CCL4L, CCL4L1 and CCL4L2 was determined in a cohort of HIV-1-uninfected individuals from the South African Black (n=23) and Caucasian (n=32) population groups using qPCR and ddPCR. A stronger correlation between the number of CCL4L copies and the sum of CCL4L1 and CCL4L2 copies generated by ddPCR (r=0.99, p<0.0001) compared to qPCR (r=0.87, p<0.0001) was observed. Real-Time qPCR exhibited greater inaccuracy at higher copy numbers which is particularly relevant to our cohort of Black individuals who have a higher range of CCL4L copies (3-6) compared to Caucasians (0-4) and a higher population median (4 and 2, respectively). Medians and ranges of CCL4L1 (Black: 2, 0-4, Caucasian: 0, 0-2) and CCL4L2 (Black: 2, 1-5, Caucasian: 2, 0-3) were also higher in the Black population. Droplet digital PCR was shown to be a far superior method to qPCR for assessment of CCL4 gene copy number variation, the accuracy of which is essential for studies of the contribution of variable gene copy number to phenotypic outcomes of host infection and disease course. PMID:24727646

  15. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri.

    PubMed

    Zhao, Yun; Xia, Qingyan; Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  16. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

    PubMed Central

    Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  17. Identification and Validation of Reference Genes for Gene Expression Analysis Using Quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)

    PubMed Central

    Gao, Xiwu; Kang, Tinghao; Zhan, Sha; Wan, Hu; Li, Jianhong

    2013-01-01

    Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms. PMID:23874494

  18. Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae).

    PubMed

    Lu, Yanhui; Yuan, Miao; Gao, Xiwu; Kang, Tinghao; Zhan, Sha; Wan, Hu; Li, Jianhong

    2013-01-01

    Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms. PMID:23874494

  19. Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

    EPA Science Inventory

    Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

  20. Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species

    PubMed Central

    Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-01-01

    Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

  1. Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species.

    PubMed

    Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-01-01

    Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

  2. Determination of the Efficacy of Two Building Decontamination Strategies by Surface Sampling with Culture and Quantitative PCR Analysis

    PubMed Central

    Buttner, Mark P.; Cruz, Patricia; Stetzenbach, Linda D.; Klima-Comba, Amy K.; Stevens, Vanessa L.; Cronin, Tracy D.

    2004-01-01

    The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus (“Bacillus subtilis subsp. niger,” also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 105 to 106 CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies. PMID:15294810

  3. Quantitation of Bt-176 maize genomic sequences by surface plasmon resonance-based biospecific interaction analysis of multiplex polymerase chain reaction (PCR).

    PubMed

    Feriotto, Giordana; Gardenghi, Sara; Bianchi, Nicoletta; Gambari, Roberto

    2003-07-30

    Surface plasmon resonance (SPR) based biosensors have been described for the identification of genetically modified organisms (GMO) by biospecific interaction analysis (BIA). This paper describes the design and testing of an SPR-based BIA protocol for quantitative determinations of GMOs. Biotinylated multiplex Polymerase Chain Reaction (PCR) products from nontransgenic maize as well as maize powders containing 0.5 and 2% genetically modified Bt-176 sequences were immobilized on different flow cells of a sensor chip. After immobilization, different oligonucleotide probes recognizing maize zein and Bt-176 sequences were injected. The results obtained were compared with Southern blot analysis and with quantitative real-time PCR assays. It was demonstrated that sequential injections of Bt-176 and zein probes to sensor chip flow cells containing multiplex PCR products allow discrimination between PCR performed using maize genomic DNA containing 0.5% Bt-176 sequences and that performed using maize genomic DNA containing 2% Bt-176 sequences. The efficiency of SPR-based BIA in discriminating material containing different amounts of Bt-176 maize is comparable to real-time quantitative PCR and much more reliable than Southern blotting, which in the past has been used for semiquantitative purposes. Furthermore, the approach allows the BIA assay to be repeated several times on the same multiplex PCR product immobilized on the sensor chip, after washing and regeneration of the flow cell. Finally, it is emphasized that the presented strategy to quantify GMOs could be proposed for all of the SPR-based, commercially available biosensors. Some of these optical SPR-based biosensors use, instead of flow-based sensor chips, stirred microcuvettes, reducing the costs of the experimentation. PMID:14705890

  4. Assessing chlorinated ethene degradation in a large scale contaminant plume by dual carbon-chlorine isotope analysis and quantitative PCR.

    PubMed

    Hunkeler, Daniel; Abe, Yumiko; Broholm, Mette M; Jeannottat, Simon; Westergaard, Claus; Jacobsen, Carsten Suhr; Aravena, Ramon; Bjerg, Poul L

    2011-01-25

    The fate of chlorinated ethenes in a large contaminant plume originating from a tetrachloroethene (PCE) source in a sandy aquifer in Denmark was investigated using novel methods including compound-specific carbon and chlorine isotope analysis and quantitative real-time polymerase chain reaction (qPCR) methods targeting Dehaloccocoides sp. and vcrA genes. Redox conditions were characterized as well based on concentrations of dissolved redox sensitive compounds and sulfur isotopes in SO(4)(2-). In the first 400 m downgradient of the source, the plume was confined to the upper 20 m of the aquifer. Further downgradient it widened in vertical direction due to diverging groundwater flow reaching a depth of up to 50 m. As the plume dipped downward and moved away from the source, O(2) and NO(3)(-) decreased to below detection levels, while dissolved Fe(2+) and SO(4)(2-) increased above detectable concentrations, likely due to pyrite oxidation as confirmed by the depleted sulfur isotope signature of SO(4)(2-). In the same zone, PCE and trichloroethene (TCE) disappeared and cis-1,2-dichloroethene (cDCE) became the dominant chlorinated ethene. PCE and TCE were likely transformed by reductive dechlorination rather than abiotic reduction by pyrite as indicated by the formation of cDCE and stable carbon isotope data. TCE and cDCE showed carbon isotope trends typical for reductive dechlorination with an initial depletion of (13)C in the daughter products followed by an enrichment of (13)C as degradation proceeded. At 1000 m downgradient of the source, cDCE was the dominant chlorinated ethene and had reached the source δ(13)C value confirming that cDCE was not affected by abiotic or biotic degradation. Further downgradient (up to 1900 m), cDCE became enriched in (13)C by up to 8 ‰ demonstrating its further transformation while vinylchloride (VC) concentrations remained low (<1 μg/L) and ethene was not observed. The correlated shift of carbon and chlorine isotope ratios of c

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

    PubMed

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

    2015-01-01

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

  6. Real-time quantitative PCR for analysis of candidate fungal biopesticides against malaria: Technique validation and first applications

    PubMed Central

    Bell, Andrew S.; Blanford, Simon; Jenkins, Nina; Thomas, Matthew B.; Read, Andrew F.

    2009-01-01

    Recent research has indicated that fungal biopesticides could augment existing malaria vector control tools. Here we present a set of methodologies to monitor the in vivo kinetics of entomopathogenic fungi in Anopheles in the presence or absence of malaria parasites using quantitative real-time PCR. Three qPCR assays were successfully developed for counting fungal genomes: “specific” assays capable of distinguishing two well characterized fungal entomopathogens Beauveria bassiana isolate IMI391510 and Metarhizium anisopliae var. acridum isolate IMI330189, both of which have previously been shown to be virulent to Anopheles mosquitoes, and a “generic” fungal assay for determining any fungal burden. A fourth assay to Plasmodium chabaudi enabled quantification of co-infecting malarial parasites. All qPCR assays provide sensitive, target-specific, and robust quantification over a linear range of greater than five orders of magnitude (seven orders of magnitude for the fungal assays). B. bassiana growth within mosquitoes exposed to three different conidial challenge doses was monitored using the B. bassiana-specific assay and represents the first description of entomopathogenic fungal replication within an insect host. This revealed that, irrespective of challenge dose, after several days of relatively little replication, a sudden on-set of substantial nuclear division occurs, accompanied by physical fungal growth (hyphae) within the mosquito haemocoel shortly before death. Exposure to higher densities of conidia resulted in significantly greater pick-up by mosquitoes and to elevated fungal burdens at each time point sampled. High fungal burdens, comparable to those identified in cadavers, were attained more rapidly and mortalities occurred earlier post-exposure with increasing challenge dose. The lines of research made possible by the qPCR assays described here will contribute to optimization of fungal biopesticides against malaria and other vector

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2012-07-01

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

  10. Analysis of DNA damage and repair in nuclear and mitochondrial DNA of animal cells using quantitative PCR

    PubMed Central

    Furda, Amy M.; Bess, Amanda Smith; Meyer, Joel N.; Van Houten, Bennett

    2015-01-01

    This chapter was written as a guide to using the long-amplicon quantitative PCR (QPCR) assay for the measurement of DNA damage in mammalian as well as non-mammalian species such as C. elegans (nematodes), Drosophila melanogaster (fruit flies) and two species of fish (Fundulus heteroclitus and Danio rerio). Since its development in the early 1990s [1-3], the QPCR assay has been widely used to measure DNA damage and repair kinetics in nuclear and mitochondrial genomes after genotoxin exposure [3-5]. One of the main strengths of the assay is that the labor-intensive and artifact-generating step of mitochondrial isolation is not needed for the accurate measurement of mtDNA copy number and damage. Below we present the advantages and limitations of using QPCR to assay DNA damage in animal cells and provide a detailed protocol of the QPCR assay that integrates its usage in newly developed animal systems. PMID:22941600

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

    PubMed Central

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

    2016-01-01

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

  12. Establishment of a sensitive system for analysis of human vaginal microbiota on the basis of rRNA-targeted reverse transcription-quantitative PCR.

    PubMed

    Kurakawa, Takashi; Ogata, Kiyohito; Tsuji, Hirokazu; Kado, Yukiko; Takahashi, Takuya; Kida, Yumi; Ito, Masahiro; Okada, Nobuhiko; Nomoto, Koji

    2015-04-01

    Ten specific primer sets, for Lactobacillus gasseri, Lactobacillus crispatus, Atopobium vaginae, Gardnerella vaginalis, Mobiluncus curtisii, Chlamydia trachomatis/muridarum, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium angulatum, were developed for quantitative analysis of vaginal microbiota. rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) analysis of the vaginal samples from 12 healthy Japanese volunteers using the new primer sets together with 25 existing primer sets revealed the diversity of their vaginal microbiota: Lactobacilli such as L. crispatus, L. gasseri, Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus vaginalis, as the major populations at 10(7) cells/ml vaginal fluid, were followed by facultative anaerobes such as Streptococcus and strict anaerobes at lower population levels of 10(4) cells/ml or less. Certain bacterial vaginosis (BV)-related bacteria, such as G. vaginalis, A. vaginae, M. curtisii, and Prevotella, were also detected in some subjects. Especially in one subject, both G. vaginalis and A. vaginae were detected at high population levels of 10(8.8) and 10(8.9) cells/ml vaginal fluid, suggesting that she is an asymptomatic BV patient. These results suggest that the RT-qPCR system is effective for accurate analysis of major vaginal commensals and diagnosis of several vaginal infections. PMID:25661498

  13. Quantitative PCR marker genes for endometrial adenocarcinoma.

    PubMed

    Kölbl, Alexandra C; Victor, Lisa-Marie; Birk, Amelie E; Jeschke, Udo; Andergassen, Ulrich

    2016-09-01

    Endometrial adenocarcinoma is a common malignancy in women worldwide, with formation of remote metastasis occurring following oncological treatment. Circulating tumor cells (CTCs) are regarded to be the origin of haematogenous metastasis formation. The present study aimed to identify suitable marker genes using a quantitative polymerase chain reaction (qPCR) approach to detect CTCs from blood samples of patients with endometrial carcinoma. Therefore, RNA was isolated from endometrial adenocarcinoma cell lines and from healthy endometrial tissue and reverse transcribed to cDNA, which was then used in qPCR on a number of marker genes. Cytokeratin 19 and claudin 4 were identified as suitable marker genes for CTCs in endometrial adenocarcinoma, due to their high expression in the majority of the cell lines investigated. The expression values of the genes examined varied widely between the different cell lines, which is similar to the variation in the patient samples. Therefore, the necessity for a set of genes for CTC detection and not one single marker gene is demonstrated. qPCR is a fast, cost‑efficient and easy to perform technique, which may be used in the detection of CTCs. Investigation of the occurrence of CTCs in cancer patients would aid in the prevention of metastasis and thereby refine treatment. PMID:27431566

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

    PubMed

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

    2014-01-01

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

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

    USGS Publications Warehouse

    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.

  16. Estimation of the genome sizes of the chigger mites Leptotrombidium pallidum and Leptotrombidium scutellare based on quantitative PCR and k-mer analysis

    PubMed Central

    2014-01-01

    Background Leptotrombidium pallidum and Leptotrombidium scutellare are the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. Before these organisms can be subjected to whole-genome sequencing, it is necessary to estimate their genome sizes to obtain basic information for establishing the strategies that should be used for genome sequencing and assembly. Method The genome sizes of L. pallidum and L. scutellare were estimated by a method based on quantitative real-time PCR. In addition, a k-mer analysis of the whole-genome sequences obtained through Illumina sequencing was conducted to verify the mutual compatibility and reliability of the results. Results The genome sizes estimated using qPCR were 191 ± 7 Mb for L. pallidum and 262 ± 13 Mb for L. scutellare. The k-mer analysis-based genome lengths were estimated to be 175 Mb for L. pallidum and 286 Mb for L. scutellare. The estimates from these two independent methods were mutually complementary and within a similar range to those of other Acariform mites. Conclusions The estimation method based on qPCR appears to be a useful alternative when the standard methods, such as flow cytometry, are impractical. The relatively small estimated genome sizes should facilitate whole-genome analysis, which could contribute to our understanding of Arachnida genome evolution and provide key information for scrub typhus prevention and mite vector competence. PMID:24947244

  17. A Comparison between Droplet Digital and Quantitative PCR in the Analysis of Bacterial 16S Load in Lung Tissue Samples from Control and COPD GOLD 2

    PubMed Central

    Sze, Marc A.; Abbasi, Meysam; Hogg, James C.; Sin, Don D.

    2014-01-01

    Background Low biomass in the bacterial lung tissue microbiome utilizes quantitative PCR (qPCR) 16S bacterial assays at their limit of detection. New technology like droplet digital PCR (ddPCR) could allow for higher sensitivity and accuracy of quantification. These attributes are needed if specific bacteria within the bacterial lung tissue microbiome are to be evaluated as potential contributors to diseases such as chronic obstructive pulmonary disease (COPD). We hypothesize that ddPCR is better at quantifying the total bacterial load in lung tissue versus qPCR. Methods Control (n = 16) and COPD GOLD 2 (n = 16) tissue samples were obtained from patients who underwent lung resection surgery, were cut on a cryotome, and sections were assigned for use in quantitative histology or for DNA extraction. qPCR and ddPCR were performed on these samples using primers spanning the V2 region on the 16S rRNA gene along with negative controls. Total 16S counts were compared between the two methods. Both methods were assessed for correlations with quantitative histology measurements of the tissue. Results There was no difference in the average total 16S counts (P>0.05) between the two methods. However, the negative controls contained significantly lower counts in the ddPCR (0.55 ± 0.28 16S/uL) than in the qPCR assay (1.00 ± 0.70 16S copies) (P <0.05). The coefficient of variation was significantly lower for the ddPCR assay (0.18 ± 0.14) versus the qPCR assay (0.62 ± 0.29) (P<0.05). Conclusion Overall the ddPCR 16S assay performed better by reducing the background noise in 16S of the negative controls compared with 16S qPCR assay. PMID:25329701

  18. Identification of Suitable Reference Genes for Gene Expression Normalization in the Quantitative Real-Time PCR Analysis of Sweet Osmanthus (Osmanthus fragrans Lour.)

    PubMed Central

    Wang, Yiguang; Bao, Zhiyi; Zhao, Hongbo

    2015-01-01

    Quantitative real-time PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Several studies examining the selection of reference genes have been performed in ornamental plants but none in sweet osmanthus (Osmanthus fragrans Lour.). Based on transcriptomic sequencing data from O. fragrans buds at four developmental stages, six reference genes (OfACT, OfEF1α, OfIDH, OfRAN1, OfTUB, and OfUBC2) with stable expression (0.5 to 2 fold change in expression levels between any two developmental stages), as well as the commonly used reference gene Of18S, were selected as candidates for gene expression normalization in the RT-qPCR analysis of O. fragrans. For the normalization of RT-qPCR with two dyes, SYBR Green and EvaGreen, the expressional stability of seven candidate reference genes in 43 O. fragrans samples was analyzed using geNorm, NormFinder and BestKeeper. For RT-qPCR using SYBR Green, OfRAN1 and OfUBC2 were the optimal reference genes for all samples and different cultivars, OfACT and OfEF1α were suitable for different floral developmental stages, and OfACT was the optimal reference gene for different temperature treatments. The geometric mean values of the optimal reference gene pairs for the normalization of RT-qPCR are recommended to be used for all samples, different cultivars and different floral developmental stages in O. fragrans. For RT-qPCR using EvaGreen, OfUBC2 was the optimal reference gene for all samples and different cultivars, and OfACT was the optimal reference gene for different floral developmental stages and different temperature treatments. As the worst reference gene, Of18S should not be used as a reference gene in O. fragrans in the future. Our results provide a reference gene application guideline for O. fragrans gene expression characterization using RT-qPCR. PMID:26302211

  19. Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea▿

    PubMed Central

    Andree, Karl B.; Fernández-Tejedor, Margarita; Elandaloussi, Laurence M.; Quijano-Scheggia, Sonia; Sampedro, Nagore; Garcés, Esther; Camp, Jordi; Diogène, Jorge

    2011-01-01

    The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy. PMID:21193668

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

    PubMed

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

    2016-09-01

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

  1. EVALUATION OF DIFFERENT METHODS FOR THE EXTRACTION OF DNA FROM FUNGAL CONIDIA BY QUANTITATIVE COMPETITIVE PCR ANALYSIS

    EPA Science Inventory

    Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartaru...

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

    PubMed Central

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

    2002-01-01

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

  3. Real-Time PCR for Quantitative Analysis of Human Commensal Escherichia coli Populations Reveals a High Frequency of Subdominant Phylogroups

    PubMed Central

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

    2013-01-01

    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., 105 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. PMID:23770894

  4. Quantitative Real-Time PCR: Recent Advances.

    PubMed

    Singh, Charanjeet; Roy-Chowdhuri, Sinchita

    2016-01-01

    Quantitative real-time polymerase chain reaction is a technique for simultaneous amplification and product quantification of a target DNA as the process takes place in real time in a "closed-tube" system. Although this technique can provide an absolute quantification of the initial template copy number, quantification relative to a control sample or second sequence is typically adequate. The quantification process employs melting curve analysis and/or fluorescent detection systems and can provide amplification and genotyping in a relatively short time. Here we describe the properties and uses of various fluorescent detection systems used for quantification. PMID:26843055

  5. Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time PCR (qPCR) such as TaqMan and SYBR Green qPCR are widely used for gene expression analysis. The drawbacks of SYBR Green assay are that the dye binds to any double-stranded DNA which can generate falsepositive signals and that the length of the amplicon affects the intensity of...

  6. The hsp 16 gene of the probiotic Lactobacillus acidophilus is differently regulated by salt, high temperature and acidic stresses, as revealed by reverse transcription quantitative PCR (qRT-PCR) analysis.

    PubMed

    Capozzi, Vittorio; Arena, Mattia Pia; Crisetti, Elisabetta; Spano, Giuseppe; Fiocco, Daniela

    2011-01-01

    Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) procedure was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3% w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group. PMID:21954366

  7. High-resolution detection of recurrent aberrations in lung adenocarcinomas by array comparative genomic hybridization and expression analysis of selective genes by quantitative PCR.

    PubMed

    Zhu, Hong; Wong, Maria Pik; Tin, Vicky

    2014-06-01

    Genomic abnormalities are the hallmark of cancers and may harbor potential candidate genes important for cancer development and progression. We performed array comparative genomic hybridization (array CGH) on 36 cases of primary lung adenocarcinoma (AD) using an array containing 2621 BAC or PAC clones spanning the genome at an average interval of 1 Mb. Array CGH identified the commonest aberrations consisting of DNA gains within 1p, 1q, 5p, 5q, 7p, 7q, 8q, 11q, 12p, 13q, 16p, 17q, 20q, and losses with 6q, 9p, 10q and 18q. High-level copy gains involved mainly 7p21-p15 and 20q13.3. Dual color fluorescence in situ hybridization (FISH) was performed on a selective locus for validation of array CGH results. Genomic aberrations were compared with different clinicopathological features and a trend of higher number of aberrations in tumors with aggressive phenotypes and current tobacco exposure was identified. According to array CGH data, 23 candidate genes were selected for quantitative PCR (qPCR) analysis. The concordance observed between the genomic and expression changes in most of the genes suggested that they could be candidate cancer-related genes that contributed to the development of lung AD. PMID:24728343

  8. Quantitative Real-Time PCR Analysis of YKL-40 and Its Comparison with Mammalian Chitinase mRNAs in Normal Human Tissues Using a Single Standard DNA

    PubMed Central

    Ohno, Misa; Bauer, Peter O.; Kida, Yuta; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

    2015-01-01

    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. PMID:25941933

  9. Comparison of Enterococcus quantitative polymerase chain reaction analysis results from midwest U.S. river samples using EPA Method 1611 and Method 1609 PCR reagents

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA) has provided recommended beach advisory values in its 2012 recreational water quality criteria (RWQC) for states wishing to use quantitative polymerase chain reaction (qPCR) for the monitoring of Enterococcus fecal indicator bacteria...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Measuring and mitigating inhibition during real-time, quantitative PCR analysis of viral nucleic acid extracts from large-volume environmental water samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Naturally-occurring inhibitory compounds are a major concern during qPCR and RT-qPCR analysis of environmental samples, particularly large volume water samples. Here, a standardized method for measuring and mitigating sample inhibition in environmental water concentrates is described. Specifically, ...

  12. Quantitative PCR for genetic markers of human fecal pollution

    EPA Science Inventory

    Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for enumeration of two recently described hum...

  13. Quantitative PCR for Genetic Markers of Human Fecal Pollution

    EPA Science Inventory

    Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantificationapproach. We report the development of quantitative PCR assays for quantification of two recently described human-...

  14. Annual distribution of allergenic fungal spores in atmospheric particulate matter in the Eastern Mediterranean; a comparative study between ergosterol and quantitative PCR analysis

    NASA Astrophysics Data System (ADS)

    Lang-Yona, N.; Dannemiller, K.; Yamamoto, N.; Burshtein, N.; Peccia, J.; Yarden, O.; Rudich, Y.

    2012-03-01

    Airborne fungal spores are an important fraction of atmospheric particulate matter and are major causative agents of allergenic and infectious diseases. Predicting the variability and species of allergy-causing fungal spores requires detailed and reliable methods for identification and quantification. There are diverse methods for their detection in the atmosphere and in the indoor environments; yet, it is important to optimize suitable methods for characterization of fungal spores in atmospheric samples. In this study we sampled and characterized total and specific airborne fungal spores from PM10 samples collected in Rehovot, Israel over an entire year. The total fungal spore concentrations vary throughout the year although the species variability was nearly the same. Seasonal equivalent spore concentrations analyzed by real-time quantitative-PCR-based methods were fall > winter > spring > summer. Reported concentrations based on ergosterol analysis for the same samples were and fall > spring > winter > summer. Correlation between the two analytical methods was found only for the spring season. These poor associations may be due to the per-spore ergosterol variations that arise from both varying production rates, as well as molecular degradation of ergosterol. While conversion of genome copies to spore concentration is not yet straightforward, the potential for improving this conversion and the ability of qPCR to identify groups of fungi or specific species makes this method preferable for environmental spore quantification. Identifying tools for establishing the relation between the presence of species and the actual ability to induce allergies is still needed in order to predict the effect on human health.

  15. Annual distribution of allergenic fungal spores in atmospheric particulate matter in the eastern mediterranean; a comparative study between ergosterol and quantitative PCR analysis

    NASA Astrophysics Data System (ADS)

    Lang-Yona, N.; Dannemiller, K.; Yamamoto, N.; Burshtein, N.; Peccia, J.; Yarden, O.; Rudich, Y.

    2011-10-01

    Airborne fungal spores are an important fraction of atmospheric particulate matter and are major causative agents of allergenic and infectious diseases. Predicting the variability and species of allergy-causing fungal spores requires detailed and reliable methods for identification and quantification. There are diverse methods for their detection in the atmosphere and in the indoor environments; yet, it is important to optimize suitable methods for characterization of fungal spores in atmospheric samples. In this study we sampled and characterized total and specific airborne fungal spores from PM10 samples collected in Rohovot, Israel over an entire year. The total fungal spore concentrations vary throughout the year although the species variability was nearly the same. Seasonal equivalent spore concentrations analyzed by real-time quantitative-PCR-based methods were fall > winter > spring > summer. Reported concentrations based on ergosterol analysis for the same samples were and fall > spring > winter > summer. Correlation between the two analytical methods was found only for the spring season. These poor associations may be due to the per-spore ergosterol variations that arise from both varying production rates, as well as molecular degradation of ergosterol. While conversion of genome copies to spore concentration is not yet straightforward, the potential for improving this conversion and the ability of qPCR to identify groups of fungi or specific species makes this method preferable for environmental spore quantification. Identifying tools for establishing the relation between the presence of species and the actual ability to induce allergies is still needed in order to predict the effect on human health.

  16. Quantitative PCR Analysis of Functional Genes in Iron-Rich Microbial Mats at an Active Hydrothermal Vent System (Lō'ihi Seamount, Hawai'i)

    PubMed Central

    Jesser, Kelsey J.; Fullerton, Heather; Hager, Kevin W.

    2015-01-01

    The chemolithotrophic Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III) and are the dominant bacterial population in iron-rich microbial mats. Zetaproteobacteria were first discovered at Lō'ihi Seamount, located 35 km southeast off the big island of Hawai'i, which is characterized by low-temperature diffuse hydrothermal venting. Novel nondegenerate quantitative PCR (qPCR) assays for genes associated with microbial nitrogen fixation, denitrification, arsenic detoxification, Calvin-Benson-Bassham (CBB), and reductive tricarboxylic acid (rTCA) cycles were developed using selected microbial mat community-derived metagenomes. Nitrogen fixation genes were not detected, but all other functional genes were present. This suggests that arsenic detoxification and denitrification processes are likely cooccurring in addition to two modes of carbon fixation. Two groups of microbial mat community types were identified by terminal restriction fragment length polymorphism (T-RFLP) and were further described based on qPCR data for zetaproteobacterial abundance and carbon fixation mode preference. qPCR variance was associated with mat morphology but not with temperature or sample site. Geochemistry data were significantly associated with sample site and mat morphology. Together, these qPCR assays constitute a functional gene signature for iron microbial mat communities across a broad array of temperatures, mat types, chemistries, and sampling sites at Lō'ihi Seamount. PMID:25681182

  17. Quantitative PCR analysis of functional genes in iron-rich microbial mats at an active hydrothermal vent system (Lō'ihi Seamount, Hawai'i).

    PubMed

    Jesser, Kelsey J; Fullerton, Heather; Hager, Kevin W; Moyer, Craig L

    2015-05-01

    The chemolithotrophic Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III) and are the dominant bacterial population in iron-rich microbial mats. Zetaproteobacteria were first discovered at Lō'ihi Seamount, located 35 km southeast off the big island of Hawai'i, which is characterized by low-temperature diffuse hydrothermal venting. Novel nondegenerate quantitative PCR (qPCR) assays for genes associated with microbial nitrogen fixation, denitrification, arsenic detoxification, Calvin-Benson-Bassham (CBB), and reductive tricarboxylic acid (rTCA) cycles were developed using selected microbial mat community-derived metagenomes. Nitrogen fixation genes were not detected, but all other functional genes were present. This suggests that arsenic detoxification and denitrification processes are likely cooccurring in addition to two modes of carbon fixation. Two groups of microbial mat community types were identified by terminal restriction fragment length polymorphism (T-RFLP) and were further described based on qPCR data for zetaproteobacterial abundance and carbon fixation mode preference. qPCR variance was associated with mat morphology but not with temperature or sample site. Geochemistry data were significantly associated with sample site and mat morphology. Together, these qPCR assays constitute a functional gene signature for iron microbial mat communities across a broad array of temperatures, mat types, chemistries, and sampling sites at Lō'ihi Seamount. PMID:25681182

  18. Evaluation of four genes in rice for their suitability as endogenous reference standards in quantitative PCR.

    PubMed

    Wang, Chong; Jiang, Lingxi; Rao, Jun; Liu, Yinan; Yang, Litao; Zhang, Dabing

    2010-11-24

    The genetically modified (GM) food/feed quantification depends on the reliable detection systems of endogenous reference genes. Currently, four endogenous reference genes including sucrose phosphate synthase (SPS), GOS9, phospholipase D (PLD), and ppi phosphofructokinase (ppi-PPF) of rice have been used in GM rice detection. To compare the applicability of these four rice reference genes in quantitative PCR systems, we analyzed the target nucleotide sequence variation in 58 conventional rice varieties from various geographic and phylogenic origins, also their quantification performances were evaluated using quantitative real-time PCR and GeNorm analysis via a series of statistical calculation to get a "M value" which is negative correlation with the stability of genes. The sequencing analysis results showed that the reported GOS9 and PLD taqman probe regions had detectable single nucleotide polymorphisms (SNPs) among the tested rice cultivars, while no SNPs were observed for SPS and ppi-PPF amplicons. Also, poor quantitative performance was detectable in these cultivars with SNPs using GOS9 and PLD quantitative PCR systems. Even though the PCR efficiency of ppi-PPF system was slightly lower, the SPS and ppi-PPF quantitative PCR systems were shown to be applicable for rice endogenous reference assay with less variation among the C(t) values, good reproducibility in quantitative assays, and the low M values by the comprehensive quantitative PCR comparison and GeNorm analysis. PMID:20961039

  19. Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.

    PubMed

    Yuan, Miao; Lu, Yanhui; Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

    2014-01-01

    The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for

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

    PubMed Central

    2014-01-01

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

  1. Identification of Valid Housekeeping Genes for Real-Time Quantitative PCR Analysis of Collapsed Lung Tissues of Neonatal Somatic Cell Nuclear Transfer-Derived Cattle.

    PubMed

    Liu, Yan; Zhang, Yunhai; Jiang, Qiuling; Rao, Man; Sheng, Zheya; Zhang, Yu; Du, Weihua; Hao, Haisheng; Zhao, Xueming; Xu, Zhe; Liu, Jianning; Zhu, Huabin

    2015-10-01

    Cloned calves produced by somatic cell nuclear transfer frequently suffer alveolar collapse as newborns. To study the underlying pathophysiological mechanisms responsible for this phenomenon, the expression profiles of numerous genes involved in lung development need to be investigated. Quantitative real-time PCR is commonly adopted in gene expression analysis. However, selection of an appropriate reference gene for normalization is critical for obtaining reliable and accurate results. Seven housekeeping genes-β-glucuronidase (GUSB), phosphoglycerate kinase 1 (PGK1), β-2-microglobolin (B2M), peptidylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA-box binding protein (TBP), and 5.8S ribosomal RNA (5.8S rRNA)-were selected and evaluated as candidates. Their gene expression levels in the collapsed lungs of deceased neonate cloned calves and normal lung derived from normal calves were assessed. The ranking of gene expression stability was estimated by the geNorm, NormFinder, and BestKeeper programs. 5.8S rRNA and PPIA were determined to be the most stable reference genes by geNorm and BestKeeper, whereas the combination of GAPDH and TBP was suggested as reference genes by NormFinder. Taking these results into account, we conclude that 5.8S rRNA and PPIA could be the most reliable reference genes for studying the genes involved in alveolar collapse. Moreover, 5.8S rRNA could be represented as a uniform reference gene in similar cases. PMID:26393896

  2. Validation of PCR methods for quantitation of genetically modified plants in food.

    PubMed

    Hübner, P; Waiblinger, H U; Pietsch, K; Brodmann, P

    2001-01-01

    For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials. PMID:11767156

  3. Citrus stubborn disease incidence determined by quantitative real time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time (q) PCR was developed for detection of Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), using the DNA binding fluorophore SYBR Green I. The primer pair, P58-3f/4r, developed based on sequences from the P58 putative adhesin multigene of the pathogen result...

  4. Quantitative Detection of Spiroplasma Citri by Real Time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a need to develop an accurate and rapid method to detect Spiroplasma citri, the causal agent of citrus stubborn disease for use in epidemiology studies. Quantitative real-time PCR was developed for detection of S. citri. Two sets of primers based on sequences from the P58 putative adhesin ...

  5. QUANTITATIVE PCR OF SELECTED ASPERGILLUS, PENICILLIUM AND PAECILOMYCES SPECIES

    EPA Science Inventory

    A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan®) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of Aspergillus, Penicillium and ...

  6. Interaction of quantitative PCR components with polymeric surfaces.

    PubMed

    Gonzalez, Asensio; Grimes, Ronan; Walsh, Edmond J; Dalton, Tara; Davies, Mark

    2007-04-01

    This study investigated the effect of exposing a polymerase chain reaction (PCR) mixture to capillary tubing of different materials and lengths, at different contact times and flow rates and the adsorption of major reaction components into the tubing wall. Using 0.5 mm ID tubing, lengths of 40 cm and residence times up to 45 min, none of the tested polymeric materials was found to affect subsequent PCR amplification. However, after exposure of the mixture to tubing lengths of 3 m or reduction of sample volume, PCR inhibition occurred, increasing with the volume to length ratio. Different flow velocities did not affect PCR yield. When the adsorption of individual PCR components was studied, significant DNA adsorption and even more significant adsorption of the fluorescent dye Sybr Green I was found. The results indicate that PCR inhibition in polymeric tubing results from adsorption of reaction components to wall surfaces, increasing substantially with tubing length or sample volume reduction, but not with contact time or flow velocities typical in dynamic PCR amplification. The data also highlight that chemical compatibility of polymeric capillaries with DNA dyes should be carefully considered for the design of quantitative microfluidic devices. PMID:17180709

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    PubMed

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-01

    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. PMID:27371921

  9. A QUANTITATIVE MODEL OF ERROR ACCUMULATION DURING PCR AMPLIFICATION

    PubMed Central

    Pienaar, E; Theron, M; Nelson, M; Viljoen, HJ

    2006-01-01

    The amplification of target DNA by the polymerase chain reaction (PCR) produces copies which may contain errors. Two sources of errors are associated with the PCR process: (1) editing errors that occur during DNA polymerase-catalyzed enzymatic copying and (2) errors due to DNA thermal damage. In this study a quantitative model of error frequencies is proposed and the role of reaction conditions is investigated. The errors which are ascribed to the polymerase depend on the efficiency of its editing function as well as the reaction conditions; specifically the temperature and the dNTP pool composition. Thermally induced errors stem mostly from three sources: A+G depurination, oxidative damage of guanine to 8-oxoG and cytosine deamination to uracil. The post-PCR modifications of sequences are primarily due to exposure of nucleic acids to elevated temperatures, especially if the DNA is in a single-stranded form. The proposed quantitative model predicts the accumulation of errors over the course of a PCR cycle. Thermal damage contributes significantly to the total errors; therefore consideration must be given to thermal management of the PCR process. PMID:16412692

  10. Comparison of TaqMan and SYBR Green qPCR methods for quantitative gene expression in tung tree tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time-PCR (qPCR) is widely used for gene expression analysis due to its large dynamic range, tremendous sensitivity, high sequence-specificity, little to no post-amplification processing, and sample throughput. TaqMan and SYBR Green qPCR are two frequently used methods. However, dir...

  11. EVALUATION OF QUANTITATIVE REAL TIME PCR FOR THE MEASUREMENT OF HELICOBATER PYLORI AT LOW CONCENTRATIONS IN DRINKING WATER

    EPA Science Inventory

    Aims: To determine the performance of a rapid, real time polymerase chain reaction (PCR) method for the detection and quantitative analysis Helicobacter pylori at low concentrations in drinking water.

    Methods and Results: A rapid DNA extraction and quantitative PCR (QPCR)...

  12. The numbers game: quantitative analysis of Neorickettsia sp. propagation through complex life cycle of its digenean host using real-time qPCR.

    PubMed

    Greiman, Stephen E; Tkach, Vasyl V

    2016-07-01

    Bacteria of the genus Neorickettsia are obligate intracellular endosymbionts of parasitic flukes (Digenea) and are passed through the entire complex life cycle of the parasite by vertical transmission. Several species of Neorickettsia are known to cause diseases in domestic animals, wildlife, and humans. Quantitative data on the transmission of the bacteria through the digenean life cycle is almost completely lacking. This study quantified for the first time the abundance of Neorickettsia within multiple stages of the life cycle of the digenean Plagiorchis elegans. Snails Lymnaea stagnalis collected from a pond in North Dakota were screened for the presence of digenean cercariae, which were subsequently tested for the presence of Neorickettsia. Three L. stagnalis were found shedding P. elegans cercariae infected with Neorickettsia. These snails were used to initiate three separate laboratory life cycles and obtain all life cycle stages for bacterial quantification. A quantitative real-time PCR assay targeting the GroEL gene was developed to enumerate Neorickettsia sp. within different stages of the digenean life cycle. The number of bacteria significantly increased throughout all stages, from eggs to adults. The two largest increases in number of bacteria occurred during the period from eggs to cercariae and from 6-day metacercariae to 48-h juvenile worms. These two periods seem to be the most important for Neorickettsia propagation through the complex digenean life cycle and maturation in the definitive host. PMID:27041341

  13. Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze)

    PubMed Central

    Hao, Xinyuan; Horvath, David P.; Chao, Wun S.; Yang, Yajun; Wang, Xinchao; Xiao, Bin

    2014-01-01

    Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions. PMID:25474086

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

    PubMed

    Nunes-Xavier, Caroline E; Pulido, Rafael

    2016-01-01

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

  15. Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.

    PubMed

    Lewis, April M; Rice, Kelly C

    2016-01-01

    Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a "two-step" qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specific genes of interest using SYBR Green-based qPCR and quantification of relative fold-change expression. PMID:25646613

  16. Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR

    PubMed Central

    Liu, Xihan; Gong, Jun

    2012-01-01

    Peritrichs are a diverse, ecologically important ciliate group usually with a complex life cycle. To date, the community of the peritrichs has been investigated by using morphology-based methods such as living observation and silver staining. Here we show a molecular approach for characterizing the diversity and quantity of free-living peritrichs in environmental samples. We newly designed four peritrich-specific primers targeting 18S rRNA genes that allow clone library construction, screening and analysis. A quantitative real-time PCR (qPCR) assay was developed to quantify peritrichs in environmental samples by using rDNA copy number as an indicator. DNA extracted from four water samples of contrasting environmental gradients was analysed. The results showed that the peritrich community was differentiated among these samples, and that the diversity decreased with the increase of water salinity. The qPCR results are consistent with the library sequence analysis in terms of quantity variations from sample to sample. The development of peritrich-specific primers, for the first time, for conventional PCR and qPCR assays, provides useful molecular tools for revealing the diversity and quantity of peritrich ciliates in environmental samples. Also, our study illustrates the potential of these molecular tools to ecological studies of other ciliate groups in diverse environments. PMID:23100023

  17. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

    PubMed Central

    Te, Shu Harn; Chen, Enid Yingru

    2015-01-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

  18. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

    PubMed

    Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

    2015-08-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

  19. Selection of Reference Genes for Expression Analysis Using Quantitative Real-Time PCR in the Pea Aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae)

    PubMed Central

    Liu, Yong; Zhou, Xuguo

    2014-01-01

    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. PMID:25423476

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR.

    PubMed

    Shuga, Joe; Zeng, Yong; Novak, Richard; Lan, Qing; Tang, Xiaojiang; Rothman, Nathaniel; Vermeulen, Roel; Li, Laiyu; Hubbard, Alan; Zhang, Luoping; Mathies, Richard A; Smith, Martyn T

    2013-09-01

    Cancers are heterogeneous and genetically unstable. New methods are needed that provide the sensitivity and specificity to query single cells at the genetic loci that drive cancer progression, thereby enabling researchers to study the progression of individual tumors. Here, we report the development and application of a bead-based hemi-nested microfluidic droplet digital PCR (dPCR) technology to achieve 'quantitative' measurement and single-molecule sequencing of somatically acquired carcinogenic translocations at extremely low levels (<10(-6)) in healthy subjects. We use this technique in our healthy study population to determine the overall concentration of the t(14;18) translocation, which is strongly associated with follicular lymphoma. The nested dPCR approach improves the detection limit to 1×10(-7) or lower while maintaining the analysis efficiency and specificity. Further, the bead-based dPCR enabled us to isolate and quantify the relative amounts of the various clonal forms of t(14;18) translocation in these subjects, and the single-molecule sensitivity and resolution of dPCR led to the discovery of new clonal forms of t(14;18) that were otherwise masked by the conventional quantitative PCR measurements. In this manner, we created a quantitative map for this carcinogenic mutation in this healthy population and identified the positions on chromosomes 14 and 18 where the vast majority of these t(14;18) events occur. PMID:23873959

  2. A quantitative PCR method to quantify ruminant DNA in porcine crude heparin.

    PubMed

    Concannon, Sean P; Wimberley, P Brett; Workman, Wesley E

    2011-01-01

    Heparin is a well-known glycosaminoglycan extracted from porcine intestines. Increased vigilance for transmissible spongiform encephalopathy in animal-derived pharmaceuticals requires methods to prevent the introduction of heparin from ruminants into the supply chain. The sensitivity, specificity, and precision of the quantitative polymerase chain reaction (PCR) make it a superior analytical platform for screening heparin raw material for bovine-, ovine-, and caprine-derived material. A quantitative PCR probe and primer set homologous to the ruminant Bov-A2 short interspersed nuclear element (SINE) locus (Mendoza-Romero et al. J. Food Prot. 67:550-554, 2004) demonstrated nearly equivalent affinities for bovine, ovine, and caprine DNA targets, while exhibiting no cross-reactivity with porcine DNA in the quantitative PCR method. A second PCR primer and probe set, specific for the porcine PRE1 SINE sequence, was also developed to quantify the background porcine DNA level. DNA extraction and purification was not necessary for analysis of the raw heparin samples, although digestion of the sample with heparinase was employed. The method exhibits a quantitation range of 0.3-3,000 ppm ruminant DNA in heparin. Validation parameters of the method included accuracy, repeatability, precision, specificity, range, quantitation limit, and linearity. PMID:21058016

  3. Rapid diagnosis of aneuploidy using segmental duplication quantitative fluorescent PCR.

    PubMed

    Kong, Xiangdong; Li, Lin; Sun, Lei; Fu, Kepeng; Long, Ju; Weng, Xunjin; Ye, Xuehe; Liu, Xinxiong; Wang, Bo; Yan, Shanhuo; Ye, Haiming; Fan, Zuqian

    2014-01-01

    The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR), for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36); trisomy 18 (n = 6); trisomy 13 (n = 4); 45, X (n = 5); 47, XXX (n = 3); 48, XXYY (n = 2); and unaffected controls (n = 40). We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use. PMID:24625828

  4. Quantitative PCR for genetic markers of human fecal pollution.

    PubMed

    Shanks, Orin C; Kelty, Catherine A; Sivaganesan, Mano; Varma, Manju; Haugland, Richard A

    2009-09-01

    Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 x 10(6) copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters. PMID:19592537

  5. Quantitative real-time PCR (qPCR) for Eimeria tenella replication — Implications for experimental refinement and animal welfare

    PubMed Central

    Nolan, Matthew J.; Tomley, Fiona M.; Kaiser, Pete; Blake, Damer P.

    2015-01-01

    The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R2 = 0.994) (p < 0.002) but not in those from day eight (after most oocyst shedding) (R2 = 0.006) (p > 0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R2 = 0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings

  6. Quantitative real-time PCR (qPCR) for Eimeria tenella replication--Implications for experimental refinement and animal welfare.

    PubMed

    Nolan, Matthew J; Tomley, Fiona M; Kaiser, Pete; Blake, Damer P

    2015-10-01

    The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R(2)=0.994) (p<0.002) but not in those from day eight (after most oocyst shedding) (R(2)=0.006) (p>0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R(2)=0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings. PMID:26141544

  7. Underwater application of quantitative PCR on an ocean mooring.

    PubMed

    Preston, Christina M; Harris, Adeline; Ryan, John P; Roman, Brent; Marin, Roman; Jensen, Scott; Everlove, Cheri; Birch, James; Dzenitis, John M; Pargett, Douglas; Adachi, Masao; Turk, Kendra; Zehr, Jonathon P; Scholin, Christopher A

    2011-01-01

    The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions. PMID:21829630

  8. Underwater Application of Quantitative PCR on an Ocean Mooring

    PubMed Central

    Preston, Christina M.; Harris, Adeline; Ryan, John P.; Roman, Brent; Marin, Roman; Jensen, Scott; Everlove, Cheri; Birch, James; Dzenitis, John M.; Pargett, Douglas; Adachi, Masao; Turk, Kendra; Zehr, Jonathon P.; Scholin, Christopher A.

    2011-01-01

    The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions. PMID:21829630

  9. Expression profiling by real-time quantitative polymerase chain reaction (RT-qPCR).

    PubMed

    Lech, Maciej; Anders, Hans-Joachim

    2014-01-01

    Real-time quantitative PCR is a variation of the standard PCR technique that is commonly used to quantify nucleic acid. However, in this technique the amount of amplified specific sequence can be quantified at each stage of the PCR cycle. If investigated sequence is present in large number of copies in particular sample, amplification product is detected already in earlier cycles; if the sequence is rare, amplification is observed in later cycles. Quantification of amplified product is acquired using fluorescent probes or fluorescent DNA-binding dyes. Accumulation of fluorescent signal can be measured by real-time PCR instruments during each of 35-45 cycwwles of the PCR reaction, which simplify the procedure by eliminating the visualization of the amplified products using gel electrophoresis. Real-time-PCR allows quantifying the amount of product already during the PCR reaction as soon as it is detectable. Correctly performed, this method may be used for precise gene expression analysis in life science, medicine, and diagnostics and has become the standard method of choice for the quantification of mRNA. However in the past few years it became obvious that real-time PCR is complex and variability of RNA templates, assay designs, inappropriate data normalization, and data interpretation may cause diverse analytical problems. PMID:24957236

  10. Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

    PubMed Central

    Yan, Zhaoping; Gao, Jinhang; Lv, Xiuhe; Yang, Wenjuan; Wen, Shilei; Tong, Huan; Tang, Chengwei

    2016-01-01

    The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α > 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis. PMID:27069927

  11. Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR

    PubMed Central

    Shuga, Joe; Zeng, Yong; Novak, Richard; Lan, Qing; Tang, Xiaojiang; Rothman, Nathaniel; Vermeulen, Roel; Li, Laiyu; Hubbard, Alan; Zhang, Luoping; Mathies, Richard A.; Smith, Martyn T.

    2013-01-01

    Cancers are heterogeneous and genetically unstable. New methods are needed that provide the sensitivity and specificity to query single cells at the genetic loci that drive cancer progression, thereby enabling researchers to study the progression of individual tumors. Here, we report the development and application of a bead-based hemi-nested microfluidic droplet digital PCR (dPCR) technology to achieve ‘quantitative’ measurement and single-molecule sequencing of somatically acquired carcinogenic translocations at extremely low levels (<10−6) in healthy subjects. We use this technique in our healthy study population to determine the overall concentration of the t(14;18) translocation, which is strongly associated with follicular lymphoma. The nested dPCR approach improves the detection limit to 1 × 10−7 or lower while maintaining the analysis efficiency and specificity. Further, the bead-based dPCR enabled us to isolate and quantify the relative amounts of the various clonal forms of t(14;18) translocation in these subjects, and the single-molecule sensitivity and resolution of dPCR led to the discovery of new clonal forms of t(14;18) that were otherwise masked by the conventional quantitative PCR measurements. In this manner, we created a quantitative map for this carcinogenic mutation in this healthy population and identified the positions on chromosomes 14 and 18 where the vast majority of these t(14;18) events occur. PMID:23873959

  12. Fast detection of deletion breakpoints using quantitative PCR.

    PubMed

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-06-16

    The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27333265

  13. Fast detection of deletion breakpoints using quantitative PCR.

    PubMed

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-01-01

    The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27560363

  14. Fast detection of deletion breakpoints using quantitative PCR

    PubMed Central

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-01-01

    Abstract The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27560363

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. MiRNA Analysis by Quantitative PCR in Preterm Human Breast Milk Reveals Daily Fluctuations of hsa-miR-16-5p

    PubMed Central

    Floris, Ilaria; Billard, Hélène; Boquien, Clair-Yves; Joram-Gauvard, Evelyne; Simon, Laure; Legrand, Arnaud; Boscher, Cécile; Rozé, Jean-Christophe; Bolaños-Jiménez, Francisco; Kaeffer, Bertrand

    2015-01-01

    Background and Aims Human breast milk is an extremely dynamic fluid containing many biologically-active components which change throughout the feeding period and throughout the day. We designed a miRNA assay on minimized amounts of raw milk obtained from mothers of preterm infants. We investigated changes in miRNA expression within month 2 of lactation and then over the course of 24 hours. Materials and Methods Analyses were performed on pooled breast milk, made by combining samples collected at different clock times from the same mother donor, along with time series collected over 24 hours from four unsynchronized mothers. Whole milk, lipids or skim milk fractions were processed and analyzed by qPCR. We measured hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-146-5p, and hsa-let-7a, d and g (all -5p). Stability of miRNA endogenous controls was evaluated using RefFinder, a web tool integrating geNorm, Normfinder, BestKeeper and the comparative ΔΔCt method. Results MiR-21 and miR-16 were stably expressed in whole milk collected within month 2 of lactation from four mothers. Analysis of lipids and skim milk revealed that miR-146b and let-7d were better references in both fractions. Time series (5H-23H) allowed the identification of a set of three endogenous reference genes (hsa-let-7d, hsa-let-7g and miR-146b) to normalize raw quantification cycle (Cq) data. We identified a daily oscillation of miR-16-5p. Perspectives Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48–72 hours. PMID:26474056

  18. Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

    PubMed Central

    2012-01-01

    Background The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. Results A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose–response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed. Conclusions The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the

  19. Validation of a quantitative PCR assay for detection and quantification of 'Candidatus Xenohaliotis californiensis'.

    PubMed

    Friedman, Carolyn S; Wight, Nate; Crosson, Lisa M; White, Samuel J; Strenge, Robyn M

    2014-04-01

    Withering syndrome (WS), a serious disease affecting abalone Haliotis spp., is caused by infection from an intracellular Rickettsia-like organism (WS-RLO). Diagnosis of the disease currently relies on a combination of histological examination and molecular methods (in situ hybridization, standard PCR, and sequence analysis). However, these techniques only provide a semi-quantitative assessment of bacterial load. We created a real-time quantitative PCR (qPCR) assay to specifically identify and enumerate bacterial loads of WS-RLO in abalone tissue, fecal, and seawater samples based on 16S rDNA gene copy numbers. The qPCR assay designed to detect DNA of the WS-RLO was validated according to standards set by the World Organisation for Animal Health. Standard curves derived from purified plasmid dilutions were linear across 7 logs of concentration, and efficiencies ranged from 90.2 to 97.4%. The limit of detection was 3 gene copies per reaction. Diagnostic sensitivity was 100% and specificity was 99.8%. The qPCR assay was robust, as evidenced by its high level of repeatability and reproducibility. This study has shown for the first time that WS-RLO DNA can be detected and quantified in abalone tissue, fecal, and seawater samples. The ability to detect and quantify RLO gene copies in a variety of materials will enable us to better understand transmission dynamics in both farmed and natural environments. PMID:24695238

  20. BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay

    PubMed Central

    2012-01-01

    Background Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay—BactQuant—for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant. Methods The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively. Conclusions The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions. PMID:22510143

  1. A RAPID METHOD FOR THE EXTRACTION OF FUNGAL DNA FROM ENVIRONMENTAL SAMPLES: EVALUATION IN THE QUANTITATIVE ANALYSIS OF MEMNONIELLA ECHINATA CONIDIA USING REAL TIME DETECTION OF PCR PRODUCTS

    EPA Science Inventory

    New technologies are creating the potential for using nucleic acid sequence detection to perform routine microbiological analyses of environmental samples. Our laboratory has recently reported on the development of a method for the quantitative detection of Stachybotrys chartarum...

  2. Quantitative RT-PCR profiling of the Rabbit Immune Response: Assessment of Acute Shigella flexneri Infection

    PubMed Central

    Schnupf, Pamela; Sansonetti, Philippe J.

    2012-01-01

    Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models. The rabbit is a widely accepted and commonly used animal model in the study of human diseases and infections by viral, fungal, bacterial and protozoan pathogens. Only a limited number of rabbit genes have, however, been analyzed by this method as the rabbit genome sequence remains unfinished. Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response. We hereby report the design of twenty-four quantitative PCR primer pairs covering common cytokines, chemoattractants, antimicrobials and enzymes for a rapid, sensitive and quantitative analysis of the rabbit immune response. Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format. This tool was used to analyze the rabbit innate immune response to infection with the human gastrointestinal pathogen Shigella flexneri. Beyond the known inflammatory mediators, we identified IL-22, IL-17A and IL-17F as highly upregulated cytokines and as first responders to infection during the innate phase of the host immune response. This set of qPCR primers also provides a convenient tool for monitoring the rabbit immune response during infection with other pathogens and other inflammatory conditions. PMID:22675469

  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

    PubMed Central

    2009-01-01

    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 costly than reference

  4. Development of Quantitative Real-time PCR Assays for Different Clades of "Candidatus Accumulibacter".

    PubMed

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

    2016-01-01

    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% (R(2) = 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. PMID:27142574

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

    PubMed

    Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

    2016-02-01

    Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis. PMID:26872627

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

    PubMed Central

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

    2016-01-01

    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. PMID:27142574

  8. Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta

    PubMed Central

    Rueda-Martínez, Carmen; Lamas, Oscar; Mataró, María José; Robledo-Carmona, Juan; Sánchez-Espín, Gemma; Jiménez-Navarro, Manuel; Such-Martínez, Miguel; Fernández, Borja

    2014-01-01

    Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples. PMID:24841551

  9. Quantitative rt-PCR analysis of uncoupling protein isoforms in mouse brain cortex: methodological optimization and comparison of expression with brown adipose tissue and skeletal muscle.

    PubMed

    Lengacher, Sylvain; Magistretti, Pierre J; Pellerin, Luc

    2004-07-01

    Uncoupling proteins (UCPs) present in the inner mitochondrial membrane are involved in uncoupling respiration from ATP synthesis. Five UCP isoforms have been identified but information about their presence and level of expression in the central nervous system remains incomplete. To determine the nature and proportion of UCP isoform mRNAs present in brain cortex, we developed and optimized a specific quantitative reverse-transcription polymerase chain reaction procedure. Optimal range of RNA concentrations to be used in the reverse-transcriptase reaction was determined. Primer design and concentration were optimized for each target gene while polymerase chain reaction efficiency was assessed for a range of reverse-transcriptase dilutions. Genomic contribution to the quantitative signal was evaluated for each isoform and minimized. Three reference genes were tested for normalization, and beta-actin was found to be the most stable among tissues. Results indicate that brain cortex contains significant amounts of all UCP mRNAs, with UCP5 and UCP4 being the most abundant, as opposed to brown adipose tissue and skeletal muscle, which predominantly express UCP1 and UCP3, respectively. These data provide a first quantitative assessment of UCP mRNA expression in mouse brain, showing the presence of all five isoforms with distinct proportions, thus suggesting specific roles in the central nervous system. PMID:15241186

  10. Quantitative Hydrocarbon Surface Analysis

    NASA Technical Reports Server (NTRS)

    Douglas, Vonnie M.

    2000-01-01

    The elimination of ozone depleting substances, such as carbon tetrachloride, has resulted in the use of new analytical techniques for cleanliness verification and contamination sampling. The last remaining application at Rocketdyne which required a replacement technique was the quantitative analysis of hydrocarbons by infrared spectrometry. This application, which previously utilized carbon tetrachloride, was successfully modified using the SOC-400, a compact portable FTIR manufactured by Surface Optics Corporation. This instrument can quantitatively measure and identify hydrocarbons from solvent flush of hardware as well as directly analyze the surface of metallic components without the use of ozone depleting chemicals. Several sampling accessories are utilized to perform analysis for various applications.

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

    PubMed

    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. PMID:27316653

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. Exogeneous controls to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative PCR (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5’-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi and P. meibomiae, two rust pathogens of soybean. Because of the extreme sensitivity of Q-PCR, ...

  14. Evaluation of Quantitative Real-Time PCR Assays for Detection of Citrus Greening

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus huanglongbing (HLB), or citrus greening, is a serious and industry-limiting disease. Preliminary diagnoses can be made through visual symptoms, and greater certainty can be achieved through quantitative real-time PCR (qPCR). Several qPCR procedures are available including those by designed by...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays

    PubMed Central

    Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J. L.; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

    2016-01-01

    Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download. PMID:26863543

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription.

    PubMed

    Lee, Jinwoo; Foong, Yee Hoon; Musaitif, Ibrahim; Tong, Tiegang; Jefcoate, Colin

    2016-07-01

    The steroidogenic acute regulatory protein (StAR) has been proposed to serve as the switch that can turn on/off steroidogenesis. We investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. We used 3' reverse primers in a single reaction to respectively quantify StAR primary (p-RNA), spliced (sp-RNA/mRNA), and extended 3' untranslated region (UTR) transcripts, which were quantitatively imaged by high-resolution fluorescence in situ hybridization (FISH). This approach delivers spatio-temporal resolution of initiation and splicing at single StAR loci, and transfers individual mRNA molecules to cytoplasmic sites. Gene expression was biphasic, initially showing slow splicing, transitioning to concerted splicing. The alternative 3.5-kb mRNAs were distinguished through the use of extended 3'UTR probes, which exhibited distinctive mitochondrial distribution. Combining quantitative PCR and FISH enables imaging of localization of RNA expression and analysis of RNA processing rates. PMID:27091298

  19. Quantitative PCR Method for Diagnosis of Citrus Bacterial Canker†

    PubMed Central

    Cubero, J.; Graham, J. H.; Gottwald, T. R.

    2001-01-01

    For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions. PMID:11375206

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

    PubMed

    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

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

  1. Virological Diagnosis of Herpes Simplex Virus 1 Esophagitis by Quantitative Real-Time PCR Assay

    PubMed Central

    Jazeron, Jean-François; Barbe, Coralie; Frobert, Emilie; Renois, Fanny; Talmud, Déborah; Brixi-Benmansour, Hedia; Brodard, Véronique; Andréoletti, Laurent; Diebold, Marie-Danièle

    2012-01-01

    Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the “gold standard.” From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 106 ± 1.1 × 108) than in histopathologically negative herpetic esophagitis (median = 3.1 × 103 ± 6.2 × 103) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 104 copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain. PMID:22170921

  2. Virological diagnosis of herpes simplex virus 1 esophagitis by quantitative real-time PCR assay.

    PubMed

    Jazeron, Jean-François; Barbe, Coralie; Frobert, Emilie; Renois, Fanny; Talmud, Déborah; Brixi-Benmansour, Hedia; Brodard, Véronique; Andréoletti, Laurent; Diebold, Marie-Danièle; Lévêque, Nicolas

    2012-03-01

    Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the "gold standard." From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain. PMID:22170921

  3. Digital PCR analysis of circulating nucleic acids.

    PubMed

    Hudecova, Irena

    2015-10-01

    Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date. PMID:25828047

  4. Development of a Quantitative PCR Assay for Thermophilic Spore-Forming Geobacillus stearothermophilus in Canned Food.

    PubMed

    Nakano, Miyo

    2015-01-01

    The thermophilic spore forming bacteria Geobacillus stearothermophilus is recognized as a major cause of spoilage in canned food. A quantitative real-time PCR assay was developed to specifically detect and quantify the species G. stearothermophilus in samples from canned food. The selected primer pairs amplified a 163-bp fragment of the 16S rRNA gene in a specific PCR assay with a detection limit of 12.5 fg of pure culture DNA, corresponding to DNA extracted from approximately 0.7 CFU/mL of G. stearothermophilus. Analysis showed that the bacterial species G. stearothermophilus was not detected in any canned food sample. Our approach presented here will be useful for tracking or quantifying species G. stearotethermophilus in canned food and ingredients. PMID:26412704

  5. Evaluation of Reference Genes for Quantitative Real-Time PCR in Songbirds

    PubMed Central

    Zinzow-Kramer, Wendy M.; Horton, Brent M.; Maney, Donna L.

    2014-01-01

    Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbird: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology. PMID:24780145

  6. Duplex Quantitative PCR Assay for Detection of Haemophilus influenzae That Distinguishes Fucose- and Protein D-Negative Strains.

    PubMed

    de Gier, Camilla; Pickering, Janessa L; Richmond, Peter C; Thornton, Ruth B; Kirkham, Lea-Ann S

    2016-09-01

    We have developed a specific Haemophilus influenzae quantitative PCR (qPCR) that also identifies fucose-negative and protein D-negative strains. Analysis of 100 H. influenzae isolates, 28 Haemophilus haemolyticus isolates, and 14 other bacterial species revealed 100% sensitivity (95% confidence interval [CI], 96% to 100%) and 100% specificity (95% CI, 92% to 100%) for this assay. The evaluation of 80 clinical specimens demonstrated a strong correlation between semiquantitative culture and the qPCR (P < 0.001). PMID:27335148

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

    PubMed

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

    2006-04-15

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

  8. Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae.

    PubMed

    Fu, Zhangwuke; He, Xiangkang; Cai, Shunfeng; Liu, Han; He, Xinyi; Li, Mingqian; Lu, Xingmeng

    2016-01-01

    Pebrine disease is the only mandatory quarantine item in sericultural production due to its destructive consequences. So far, the mother moth microscopic examination method established by Pasteur (1870) remains the only detection method for screening for the causative agent Nosema bombycis (N. bombycis). Because pebrine is a horizontal and vertical transmission disease, it is better to inspect silkworm eggs and newly hatched larvae to investigate the infection rate, vertical transmission rate and spore load of the progenies. There is a rising demand for a more direct, effective and accurate detection approach in the sericultural industry. Here, we developed a molecular detection approach based on real-time quantitative PCR (qPCR) for pebrine inspection in single silkworm eggs and newly hatched larvae. Targeting the small-subunit rRNA gene of N. bombycis, this assay showed high sensitivity and reproducibility. Ten spores in a whole sample or 0.1 spore DNA (1 spore DNA represents the DNA content of one N. bombycis spore) in a reaction system was estimated as the detection limit of the isolation and real-time qPCR procedure. Silkworm egg tissues impact the detection sensitivity but are not significant in single silkworm egg detection. Of 400 samples produced by infected moths, 167 and 195 were scored positive by light microscopy and real-time qPCR analysis, respectively. With higher accuracy and the potential capability of high-throughput screening, this method is anticipated to be adaptable for pebrine inspection and surveillance in the sericultural industry. In addition, this method can be applied to ecology studies of N. bombycis-silkworm interactions due to its quantitative function. PMID:26658327

  9. Establishment and evaluation of event-specific quantitative PCR method for genetically modified soybean MON89788.

    PubMed

    Takabatake, Reona; Onishi, Mari; Koiwa, Tomohiro; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Furui, Satoshi; Kitta, Kazumi

    2010-01-01

    A novel real-time PCR-based analytical method was established for the event-specific quantification of a GM soybean event MON89788. The conversion factor (C(f)) which is required to calculate the GMO amount was experimentally determined. The quantitative method was evaluated by a single-laboratory analysis and a blind test in a multi-laboratory trial. The limit of quantitation for the method was estimated to be 0.1% or lower. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were both less than 20%. These results suggest that the established method would be suitable for practical detection and quantification of MON89788. PMID:21071908

  10. OPPORTUNISTIC ASPERGILLUS PATHOGENS MEASURED IN HOME AND HOSPITAL TAP WATER BY MOLD SPECIFIC QUANTITATIVE PCR (MSQPCR)

    EPA Science Inventory

    Opportunistic fungal pathogens are a concern because of the increasing number of immunocompromised patients. The goal of this research was to test a simple extraction method and rapid quantitative PCR (QPCR) measurement of the occurrence of potential pathogens, Aspergillus fumiga...

  11. Identification of reference genes for real-time quantitative PCR experiments in the liverwort Marchantia polymorpha.

    PubMed

    Saint-Marcoux, Denis; Proust, Hélène; Dolan, Liam; Langdale, Jane A

    2015-01-01

    Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data. PMID:25798897

  12. Identification of Reference Genes for Real-Time Quantitative PCR Experiments in the Liverwort Marchantia polymorpha

    PubMed Central

    Dolan, Liam; Langdale, Jane A.

    2015-01-01

    Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data. PMID:25798897

  13. PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples

    SciTech Connect

    Wang, Rong-Fu; Cao, Wei-Wen; Cerniglia, C.E.

    1996-04-01

    PCR procedures based on 16S rRNA genen sequence specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human feces and animal feces. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps.

  14. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    EPA Science Inventory

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  15. Assessing the Validity of Diagnostic Quantitative PCR Assays for Phakopsora pachyrhizi and P. meibomiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are 123 confirmed species in the genus Phakopsora worldwide, with 19 species reported in the continental United States. In 2002, a quantitative PCR (qPCR) diagnostic assay was developed by Frederick et al. that has been used for detecting Phakopsora pachyrhizi in spore trapping studies. Based ...

  16. Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

    PubMed Central

    Guilbaud, Morgan; de Coppet, Pierre; Bourion, Fabrice; Rachman, Cinta; Prévost, Hervé; Dousset, Xavier

    2005-01-01

    A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 102 CFU/cm2. PMID:15812058

  17. Single Laboratory Comparison of Quantitative Real-time PCR Assays for the Detection of Fecal Pollution

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) assays available to detect and enumerate fecal pollution in ambient waters. Each assay employs distinct primers and probes that target different rRNA genes and microorganisms leading to potential variations in concentration es...

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

    PubMed Central

    2013-01-01

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

  19. Quantitative environmental risk analysis

    SciTech Connect

    Klovning, J.; Nilsen, E.F.

    1995-12-31

    According to regulations relating to implementation and rise of risk analysis in the petroleum activities issued by the Norwegian Petroleum Directorate, it is mandatory for an operator on the Norwegian Continental Shelf to establish acceptance criteria for environmental risk in the activities and carry out environmental risk analysis. This paper presents a {open_quotes}new{close_quotes} method for environmental risk analysis developed by the company. The objective has been to assist the company to meet rules and regulations and to assess and describe the environmental risk in a systematic manner. In the environmental risk analysis the most sensitive biological resource in the affected area is used to assess the environmental damage. The analytical method is based on the methodology for quantitative risk analysis related to loss of life. In addition it incorporates the effect of seasonal fluctuations in the environmental risk evaluations. The paper is describing the function of the main analytical sequences exemplified through an analysis of environmental risk related to exploration drilling in an environmental sensitive area on the Norwegian Continental Shelf.

  20. On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform.

    PubMed

    Tachibana, Hiroaki; Saito, Masato; Shibuya, Shogo; Tsuji, Koji; Miyagawa, Nobuyuki; Yamanaka, Keiichiro; Tamiya, Eiichi

    2015-12-15

    Polymerase chain reaction (PCR)-based genetic testing has become a routine part of clinical diagnoses and food testing. In these fields, rapid, easy-to-use, and cost-efficient PCR chips are expected to be appeared for providing such testing on-site. In this study, a new autonomous disposable plastic microfluidic PCR chip was created, and was utilized for quantitative detection of pathogenic microorganisms. To control the capillary flow of the following solution in the PCR microchannel, a driving microchannel was newly designed behind the PCR microchannel. This allowed the effective PCR by simply dropping the PCR solution onto the inlet without any external pumps. In order to achieve disposability, injection-molded cyclo-olefin polymer (COP) of a cost-competitive plastic was used for the PCR chip. We discovered that coating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface for driving the capillary flow through the 1250-mm long microchannel. As a result, quantitative real-time PCR with the lowest initial concentration of human, Escherichia coli (E. coli), and pathogenic E. coli O157 genomic DNA of 4, 0.0019, 0.031 pg/μl, respectively, was successfully achieved in less than 18 min. Our results indicate that the platform presented in this study provided a rapid, easy-to-use, and low-cost real-time PCR system that could be potentially used for on-site gene testing. PMID:26210470

  1. Quantitative genotyping of mouse brain-specific PEX13 gene disruption by real-time PCR.

    PubMed

    Müller, C Catharina; Nourse, Jamie P; Nguyen, Tam H; Crane, Denis I

    2009-06-30

    The Cre/loxP-system has become an invaluable tool for the generation of tissue-specific gene disruption in mice. However, because Cre recombinase excision of individual genes can be variable, an accurate and sensitive method is necessary to determine the ultimate level of gene disruption. The analysis of gene disruption is particularly difficult for tissue that has been fixed for (immuno)histochemical analysis with paraformaldehyde. Here, we describe a simple, rapid and cost effective method for measurement of gene disruption using quantitative real-time PCR, through application to the analysis of PEX13 gene disruption in a brain-specific PEX13 mouse mutant. We show that this general protocol is suitable for both normal and paraformaldehyde-fixed tissue. PMID:19422853

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

    PubMed Central

    Descours, G.; Tellini, C.; Flamens, C.; Philit, F.; Celard, M.; Etienne, J.; Lina, G.; Jarraud, S.

    2013-01-01

    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. PMID:23862082

  3. Nested-PCR and TaqMan real-time quantitative PCR assays for human adenoviruses in environmental waters.

    PubMed

    Huang, Wen-Chien; Chou, Yi-Pen; Kao, Po-Min; Hsu, Tsui-Kang; Su, Hung-Chang; Ho, Ying-Ning; Yang, Yi-Chun; Hsu, Bing-Mu

    2016-01-01

    Human adenovirus (HAdV) infections can occur throughout the year. Cases of HAdV-associated respiratory disease have been more common in the late winter, spring, and early summer. In this study, to provide viral pollution data for further epidemiological studies and governmental actions, the presence of HAdV in the aquatic environment was quantitatively surveyed in the summer. This study was conducted to compare the efficiencies of nested-PCR (polymerase chain reaction) and qPCR (quantitative PCR) for detecting HAdV in environmental waters. A total of 73 water samples were collected from Puzi River in Taiwan and subjected to virus concentration methods. In the results, qPCR had much better efficiency for specifying the pathogen in river sample. HAdV41 was detected most frequently in the river water sample (10.9%). The estimated HAdV concentrations ranged between 6.75 × 10(2) and 2.04 × 10(9) genome copies/L. Significant difference was also found in heterotrophic plate counts, conductivity, water temperature, and water turbidity between presence/absence of HAdV. HAdV in the Puzi River may pose a significant health risk. PMID:27120637

  4. Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation

    PubMed Central

    Wei, Yongcheng; Liu, Qinghua; Dong, Hongyu; Zhou, Zhichun; Hao, Yanping; Chen, Xuelian; Xu, Liuyi

    2016-01-01

    Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana. PMID:26800152

  5. Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree.

    PubMed

    Long, Xiangyu; He, Bin; Gao, Xinsheng; Qin, Yunxia; Yang, Jianghua; Fang, Yongjun; Qi, Jiyan; Tang, Chaorong

    2015-06-01

    In rubber tree, latex regeneration is one of the decisive factors influencing the rubber yield, although its molecular regulation is not well known. Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of latex regeneration. However, the suitable reference genes required for qPCR are not available to investigate the expressions of target genes during latex regeneration. In this study, 20 candidate reference genes were selected and evaluated for their expression stability across the samples during the process of latex regeneration. All reference genes showed a relatively wide range of the threshold cycle values, and their stability was validated by four different algorithms (comparative delta Ct method, Bestkeeper, NormFinder and GeNorm). Three softwares (comparative delta Ct method, NormFinder and GeNorm) exported similar results that identify UBC4, ADF, UBC2a, eIF2 and ADF4 as the top five suitable references, and 18S as the least suitable one. The application of the screened references would improve accuracy and reliability of gene expression analysis in latex regeneration experiments. PMID:25791491

  6. Opportunistic pathogens in roof-captured rainwater samples, determined using quantitative PCR.

    PubMed

    Ahmed, W; Brandes, H; Gyawali, P; Sidhu, J P S; Toze, S

    2014-04-15

    In this study, quantitative PCR (qPCR) was used for the detection of four opportunistic bacterial pathogens in water samples collected from 72 rainwater tanks in Southeast Queensland, Australia. Tank water samples were also tested for fecal indicator bacteria (Escherichia coli and Enterococcus spp.) using culture-based methods. Among the 72 tank water samples tested, 74% and 94% samples contained E. coli and Enterococcus spp., respectively, and the numbers of E. coli and Enterococcus spp. in tank water samples ranged from 0.3 to 3.7 log₁₀ colony forming units (CFU) per 100 mL of water. In all, 29%, 15%, 13%, and 6% of tank water samples contained Aeromonas hydrophila, Staphylococcus aureus, Pseudomonas aeruginosa and Legionella pneumophila, respectively. The genomic units (GU) of opportunistic pathogens in tank water samples ranged from 1.5 to 4.6 log₁₀ GU per 100 mL of water. A significant correlation was found between E. coli and Enterococcus spp. numbers in pooled tank water samples data (Spearman's rs = 0.50; P < 0.001). In contrast, fecal indicator bacteria numbers did not correlate with the presence/absence of opportunistic pathogens tested in this study. Based on the results of this study, it would be prudent, to undertake a Quantitative Microbial Risk Assessment (QMRA) analysis of opportunistic pathogens to determine associated health risks for potable and nonpotable uses of tank water. PMID:24531256

  7. Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods

    EPA Science Inventory

    Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an optionn for recreational water quality testi...

  8. Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

    PubMed Central

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

  9. Quantitative nucleic acid amplification by digital PCR for clinical viral diagnostics.

    PubMed

    Zhang, Kuo; Lin, Guigao; Li, Jinming

    2016-09-01

    In the past few years, interest in the development of digital PCR (dPCR) as a direct nucleic acid amplification technique for clinical viral diagnostics has grown. The main advantages of dPCR over qPCR include: quantification of nucleic acid concentrations without a calibration curve, comparable sensitivity, superior quantitative precision, greater resistance to perturbations by inhibitors, and increased robustness to the variability of the target sequence. In this review, we address the application of dPCR to viral nucleic acid quantification in clinical applications and for nucleic acid quantification standardization. Further development is required to overcome the current limitations of dPCR in order to realize its widespread use for viral load measurements in clinical diagnostic applications. PMID:26845722

  10. Development and validation of a quantitative PCR assay for Ichthyophonus spp.

    PubMed

    White, Vanessa C; Morado, J Frank; Crosson, Lisa M; Vadopalas, Brent; Friedman, Carolyn S

    2013-04-29

    Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus. PMID:23670081

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

    NASA Astrophysics Data System (ADS)

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

    2008-10-01

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

  12. High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

    PubMed Central

    2011-01-01

    Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics. PMID:22035192

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

    PubMed

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

    2016-09-01

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

  14. Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence.

    PubMed Central

    Erhardt, A; Schaefer, S; Athanassiou, N; Kann, M; Gerlich, W H

    1996-01-01

    We have developed a sensitive and quantitative assay for hepatitis B virus (HBV) DNA in serum or plasma in which PCR and then microtiter hybridization analysis are used. Assay of HBV DNA in serum or plasma is important for demonstrating viral replication, indicating and monitoring antiviral therapy, determining the infectivities of virus carriers, and ensuring the safety of blood products. Under optimum conditions PCR can amplify one HBV DNA molecule to 10(8) copies, but detection of this amount of DNA still requires hybridization with labelled probes or a nested PCR. We labelled one strand of the PCR product with a biotinylated primer. The double-stranded amplicon was incubated in streptavidin-coated microplate wells. The nonlabelled strand was removed after denaturation of the double-stranded DNA with alkali, and the bound strand was hybridized with a peroxidase-coupled single-stranded probe. The amount of bound peroxidase was measured in a luminometer. Four picograms of amplicon was detectable in this system, whereas conventional ethidium bromide staining requires a 1,000 times higher amplicon concentration. The performance of the new assay was compared with those of nested PCR and a PCR system that uses a digoxigenin label, hybridization to a solid-phase adsorbed probe, and colorimetric detection. The chemiluminescence assay was found to be almost as sensitive as nested PCR and approximately five times more sensitive than the colorimetric test. PMID:8818875

  15. [Rapid detection of Pseudomonas aeruginosa by the fluorescence quantitative PCR assay targeting 16S rDNA].

    PubMed

    Xue, Li-Jun; Wang, Yong-Zhi; Ren, Hao; Tong, Yi-Min; Zhao, Ping; Zhu, Shi-Ying; Qi, Zhong-Tian

    2006-09-01

    The 16S rDNA specific primers were designed for rapid detection of Pseudomonas aeruginosa (PA) by the fluorescence quantitative PCR (FQ-PCR) assay, based upon multiple sequence alignment and phylogenetic tree analysis of the 16S rDNAs of over 20 bacteria. After extraction of PA genomic DNA, the target 16S rDNA fragment was amplified by PCR with specific primers, and used to construct recombinant pMDT-Pfr plasmid, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay. Different concentrations of PA genomic DNA were detected by FQ-PCR in a 20microL of reaction system with SYBR Green I. At the same time, various genomic DNAs of Staphylococcus aureus, Salmonella typhi, Shigella flexneri, Proteus vulgaris, Staphylococcus epidermidis, Escherichia coli, and Mycobacterium tuberculosis were used as negative controls to confirm specificity of the FQ-PCR detection assay. Results demonstrated that the predicted amplified product of designed primers was of high homology only with PA 16S rDNA, and that sensitivity of the FQ-PCR assay was of 3.6pg/microL of bacterial DNA or (2.1 x 10(3) +/- 3.1 x 10(2)) copies/microL of 16S rDNA, accompanied with high specificity, and that the whole detection process including DNA extraction could be completed in about two hours. In contrast to traditional culture method, the FQ-PCR assay targeting 16S rDNA gene can be used to detect PA rapidly, which exhibits perfect application prospect in future. PMID:17037203

  16. Rapid and Inexpensive Screening of Genomic Copy Number Variations Using a Novel Quantitative Fluorescent PCR Method

    PubMed Central

    Han, Joan C.; Elsea, Sarah H.; Pena, Heloísa B.; Pena, Sérgio Danilo Junho

    2013-01-01

    Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR) was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations. PMID:24288428

  17. Quantitative real-time PCR (qPCR)--based tool for detection and quantification of Cordyceps militaris in soil.

    PubMed

    Saragih, Syaiful Amri; Takemoto, S; Hisamoto, Y; Fujii, M; Sato, H; Kamata, N

    2015-01-01

    A quantitative real-time PCR using a primer pair CM2946F/CM3160R was developed for specific detection and quantification of Cordyceps militaris from soil. Standard curves were obtained for genomic DNA and DNA extracts from autoclaved soil with a certain dose of C. militaris suspension. C. militaris was detected from two forest soil samples out of ten that were collected when fruit bodies of C. militaris were found. This method seemed effective in detection of C. militaris in the soil and useful for rapid and reliable quantification of C. militaris in different ecosystems. PMID:25446034

  18. Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

    PubMed

    Teferedegne, Belete; Lewis, Andrew M; Peden, Keith; Murata, Haruhiko

    2013-01-01

    A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours). In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID(50)). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses. PMID:23437084

  19. Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations.

    PubMed

    Kraytsberg, Yevgenya; Khrapko, Konstantin

    2005-09-01

    A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future. PMID:16149882

  20. Selection of Reference Genes for Quantitative Real-Time PCR in Bamboo (Phyllostachys edulis)

    PubMed Central

    Fan, Chunjie; Ma, Jinmin; Guo, Qirong; Li, Xiaotie; Wang, Hui; Lu, Mengzhu

    2013-01-01

    Background The Moso bamboo (Phyllostachys edulis) is one of the most important forestry resources and plays essential ecological roles in southern China. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression data related to the unique traits of Moso bamboo will undoubtedly follow. Reverse transcription quantitative real-time PCR ((RT-)qPCR) is a widely used method for gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. Result In this study, 14 candidate reference genes were chosen, and their expression levels were assessed by (RT-)qPCR in a set of six tissue samples (root, stem, mature stem, leaf, flower, and leaf sheath) and at two developmental stages (before and after flowering) in bamboo specimens obtained in three locations. The stability and suitability of the candidate reference genes were validated using the geNorm, NormFinder and BestKeeper programs. The results showed that TIP41 and NTB were suitable reference genes across all the tissues and at the different developmental stages examined in this study. While the expression of the NTB, TIP41 and UBQ were the mostly stable in different plant tissues samples, the expression of the TIP41, NTB and CAC were ranked the most stable in bamboo plants at various developmental stages. AP2-like gene was further assessed by using the reference genes TIP41 and NTB in comparison to ACT. Significant difference of the expression profile of AP2-like demonstrated the importance of choosing adequate reference genes in bamboo. Conclusion TIP41 and NTB were found to be homogeneously expressed and were adequate for normalization purposes, showing equivalent transcript levels in different samples. They are therefore the recommended reference genes for measuring gene expression in P. edulis. PMID:23437174

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

    PubMed Central

    2012-01-01

    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 <10% and <20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection. 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. PMID:23136846

  2. New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

    PubMed Central

    Gueimonde, Miguel; Tölkkö, Satu; Korpimäki, Teemu; Salminen, Seppo

    2004-01-01

    The application of a real-time quantitative PCR method (5′ nuclease assay), based on the use of a probe labeled at its 5′ end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 × 104 cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces. PMID:15240297

  3. [Selective detection of viable pathogenic bacteria in water using reverse transcription quantitative PCR].

    PubMed

    Lin, Yi-Wen; Li, Dan; Wu, Shu-Xu; He, Miao; Yang, Tian

    2012-11-01

    A reverse transcription q quantitative PCR (RT-qPCR) assay method was established, which can quantify the copy numbers of RNA in pathogenic bacteria of E. coli and Enterococcus faecium. The results showed that cDNA was generated with the RT-PCR reagents, target gene was quantified with the qPCR, the copy numbers of RNA were stable at about 1 copies x CFU(-1) for E. coli and 7.98 x 10(2) copies x CFU(-1) for Enterococcus faecium respectively during the stationary grow phase for the both indicator bacteria [E. coli (6-18 h) and Enterococcus faecium (10-38 h)]. The established RT-qPCR method can quantify the numbers of viable bacteria through detecting bacterial RNA targets. Through detecting the heat-treated E. coli and Enterococcus faecium by three methods (culture method, qPCR, RT-qPCR), we found that the qPCR and RT-qPCR can distinguish 1.43 lg copy non-viable E. coli and 2.5 lg copy non-viable Enterococcus faecium. These results indicated that the established methods could effectively distinguish viable bacteria from non-viable bacteria. Finally we used this method to evaluate the real effluents of the secondary sedimentation of wastewater treatment plant (WWTP), the results showed that the correlation coefficients (R2) between RT-qPCR and culture method were 0.930 (E. coli) and 0.948 (Enterococcus faecium), and this established RT-PCR method can rapidly detect viable pathogenic bacteria in genuine waters. PMID:23323443

  4. Trichothecene Genotypes of the Fusarium graminearum Species Complex Isolated from Brazilian Wheat Grains by Conventional and Quantitative PCR

    PubMed Central

    Tralamazza, Sabina M.; Braghini, Raquel; Corrêa, Benedito

    2016-01-01

    We compared two well-established methods, fungal isolation followed by conventional PCR and DNA analysis by quantitative PCR (qPCR), to define trichothecene genotypes in Brazilian wheat grains from different locations. For this purpose, after fungal isolation from 75 wheat samples, 100 isolates of the Fusarium graminearum species complex (FGSC) were genotyped by PCR to establish their trichothecene profile. For profiling by qPCR, DNA was extracted from the wheat samples and analyzed. The methods provided similar and divergent results. The FGSC isolates were classified as NIV (55%), 15-ADON (43%), and 3-ADON (2%). Analysis by qPCR showed 100% contamination with 15-ADON strains in all wheat samples, 80% contamination with the NIV genotype, and only 33.3% contamination with 3-ADON strains. Further analysis revealed that 96% of all quantified DNA was attributed to the 15-ADON profile, while 3.4% was attributed to NIV and only 0.06% to 3-ADON. A positive correlation was observed between 15-ADON genotype DNA concentration and deoxynivalenol (DON) content in the wheat samples. The high frequency of fungi, DNA levels and positive correlation with DON strongly indicate that 15-ADON producers are the main trichothecene genotype in Brazilian wheat grains. Surprisingly, although many isolates (55%) carried the NIV genotype and this genotype was identified in 80% of the wheat samples, only 3.4% of fungal DNA was in fact from NIV producers. Although, our findings showed that each method provided a different perspective about the trichothecene profile, DNA analysis by qPCR gave us new insight about fungal contamination levels in Brazilian wheat grains. Nevertheless, both techniques should be used to obtain more robust results. PMID:26973624

  5. Trichothecene Genotypes of the Fusarium graminearum Species Complex Isolated from Brazilian Wheat Grains by Conventional and Quantitative PCR.

    PubMed

    Tralamazza, Sabina M; Braghini, Raquel; Corrêa, Benedito

    2016-01-01

    We compared two well-established methods, fungal isolation followed by conventional PCR and DNA analysis by quantitative PCR (qPCR), to define trichothecene genotypes in Brazilian wheat grains from different locations. For this purpose, after fungal isolation from 75 wheat samples, 100 isolates of the Fusarium graminearum species complex (FGSC) were genotyped by PCR to establish their trichothecene profile. For profiling by qPCR, DNA was extracted from the wheat samples and analyzed. The methods provided similar and divergent results. The FGSC isolates were classified as NIV (55%), 15-ADON (43%), and 3-ADON (2%). Analysis by qPCR showed 100% contamination with 15-ADON strains in all wheat samples, 80% contamination with the NIV genotype, and only 33.3% contamination with 3-ADON strains. Further analysis revealed that 96% of all quantified DNA was attributed to the 15-ADON profile, while 3.4% was attributed to NIV and only 0.06% to 3-ADON. A positive correlation was observed between 15-ADON genotype DNA concentration and deoxynivalenol (DON) content in the wheat samples. The high frequency of fungi, DNA levels and positive correlation with DON strongly indicate that 15-ADON producers are the main trichothecene genotype in Brazilian wheat grains. Surprisingly, although many isolates (55%) carried the NIV genotype and this genotype was identified in 80% of the wheat samples, only 3.4% of fungal DNA was in fact from NIV producers. Although, our findings showed that each method provided a different perspective about the trichothecene profile, DNA analysis by qPCR gave us new insight about fungal contamination levels in Brazilian wheat grains. Nevertheless, both techniques should be used to obtain more robust results. PMID:26973624

  6. Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation

    PubMed Central

    Joly, Philippe; Falconnet, Pierre-Alain; André, Janine; Weill, Nicole; Reyrolle, Monique; Vandenesch, François; Maurin, Max; Etienne, Jerome; Jarraud, Sophie

    2006-01-01

    Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >103 CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory. PMID:16597985

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

    PubMed

    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

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

    PubMed Central

    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

  9. Assessment of Soil Microbial Community Structure by Use of Taxon-Specific Quantitative PCR Assays

    PubMed Central

    Fierer, Noah; Jackson, Jason A.; Vilgalys, Rytas; Jackson, Robert B.

    2005-01-01

    Here we describe a quantitative PCR-based approach to estimating the relative abundances of major taxonomic groups of bacteria and fungi in soil. Primers were thoroughly tested for specificity, and the method was applied to three distinct soils. The technique provides a rapid and robust index of microbial community structure. PMID:16000830

  10. DETECTION OF RENIBACTERIUM SALMONINARUM IN CHINOOK SALMON ONCORHYNCHUS TSHAWYTSCHA USING QUANTITATIVE PCR.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed a quantitative PCR assay to detect varying levels of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). This assay allows for the direct enumeration of bacterial DNA or RNA copy number within tissues and body fluids. The assay can be applied nonletha...

  11. DETECTION AND QUANTITATION OF SOLENOPSIS INVICTA VIRUS IN FIRE ANTS BY REAL-TIME PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of Solenopsis invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) f...

  12. A Multiplexed, Probe-Based Quantitative PCR Assay for DNA of Phytophthora sojae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytophthora sojae (Kaufm. & Gerd.) causes seed rot, pre- and post-emergence damping off, and sometimes foliar blight in soybean (Glycine max). Crop loss may approach 100% with susceptible cultivars. We report here the development of a unique quantitative PCR assay specific to DNA of P. sojae, and a...

  13. Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay.

    PubMed

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-05-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

  14. Rapid Staphylococcus aureus agr Type Determination by a Novel Multiplex Real-Time Quantitative PCR Assay

    PubMed Central

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-01-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

  15. Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

    EPA Science Inventory

    Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described cow feces-spec...

  16. Application of quantitative PCR assays to detection of human Bacteroides species in the intestines of pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    When evaluating the efficacy of probiotic bacteria, it is beneficial to know whether a fed bacterium reaches the appropriate location in the digestive tract. Use of quantitative PCR could detect specific bacteria in a sample with a complex microbial community. In order to determine whether three Bac...

  17. Quantitative Assessment of Commutability for Clinical Viral Load Testing Using a Digital PCR-Based Reference Standard.

    PubMed

    Tang, L; Sun, Y; Buelow, D; Gu, Z; Caliendo, A M; Pounds, S; Hayden, R T

    2016-06-01

    Given recent advances in the development of quantitative standards, particularly WHO international standards, efforts to better understand the commutability of reference materials have been made. Existing approaches in evaluating commutability include prediction intervals and correspondence analysis; however, the results obtained from existing approaches may be ambiguous. We have developed a "deviation-from-ideal" (DFI) approach to evaluate commutability of standards and applied it to the assessment of Epstein-Bar virus (EBV) load testing in four quantitative PCR assays, treating digital PCR as a reference assay. We then discuss advantages and limitations of the DFI approach as well as experimental design to best evaluate the commutability of an assay in practice. PMID:27076654

  18. Validation of reference genes for real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.

    PubMed

    Zhao, Wenjing; Li, Yan; Gao, Pengfei; Sun, Zhihong; Sun, Tiansong; Zhang, Heping

    2011-09-01

    Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang. PMID:21104423

  19. Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

    USGS Publications Warehouse

    Purcell, Maureen K.; Getchell, Rodman G.; McClure, Carol A.; Weber, S.E.; Garver, Kyle A.

    2011-01-01

    Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

  20. The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces.

    PubMed

    Ståhl, M; Kokotovic, B; Hjulsager, C K; Breum, S Ø; Angen, Ø

    2011-08-01

    Four quantitative PCR (qPCR) assays were evaluated for quantitative detection of Brachyspira pilosicoli, Lawsonia intracellularis, and E. coli fimbrial types F4 and F18 in pig feces. Standard curves were based on feces spiked with the respective reference strains. The detection limits from the spiking experiments were 10(2) bacteria/g feces for Bpilo-qPCR and Laws-qPCR, 10(3)CFU/g feces for F4-qPCR and F18-qPCR. The PCR efficiency for all four qPCR assays was between 0.91 and 1.01 with R(2) above 0.993. Standard curves, slopes and elevation, varied between assays and between measurements from pure DNA from reference strains and feces spiked with the respective strains. The linear ranges found for spiked fecal samples differed both from the linear ranges from pure culture of the reference strains and between the qPCR tests. The linear ranges were five log units for F4-qPCR, and Laws-qPCR, six log units for F18-qPCR and three log units for Bpilo-qPCR in spiked feces. When measured on pure DNA from the reference strains used in spiking experiments, the respective log ranges were: seven units for Bpilo-qPCR, Laws-qPCR and F18-qPCR and six log units for F4-qPCR. This shows the importance of using specific standard curves, where each pathogen is analysed in the same matrix as sample DNA. The qPCRs were compared to traditional bacteriological diagnostic methods and found to be more sensitive than cultivation for E. coli and B. pilosicoli. The qPCR assay for Lawsonia was also more sensitive than the earlier used method due to improvements in DNA extraction. In addition, as samples were not analysed for all four pathogen agents by traditional diagnostic methods, many samples were found positive for agents that were not expected on the basis of age and case history. The use of quantitative PCR tests for diagnosis of enteric diseases provides new possibilities for veterinary diagnostics. The parallel simultaneous analysis for several bacteria in multi-qPCR and the

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

    PubMed Central

    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. PMID:27242878

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

    PubMed

    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. PMID:27242878

  3. Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

    EPA Science Inventory

    Purified oocysts of Cryptosporidium parvum were used to evaluate applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-...

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

    PubMed Central

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

    2007-01-01

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

  5. Comparative Application of PLS and PCR Methods to Simultaneous Quantitative Estimation and Simultaneous Dissolution Test of Zidovudine - Lamivudine Tablets.

    PubMed

    Üstündağ, Özgür; Dinç, Erdal; Özdemir, Nurten; Tilkan, M Günseli

    2015-01-01

    In the development strategies of new drug products and generic drug products, the simultaneous in-vitro dissolution behavior of oral dosage formulations is the most important indication for the quantitative estimation of efficiency and biopharmaceutical characteristics of drug substances. This is to force the related field's scientists to improve very powerful analytical methods to get more reliable, precise and accurate results in the quantitative analysis and dissolution testing of drug formulations. In this context, two new chemometric tools, partial least squares (PLS) and principal component regression (PCR) were improved for the simultaneous quantitative estimation and dissolution testing of zidovudine (ZID) and lamivudine (LAM) in a tablet dosage form. The results obtained in this study strongly encourage us to use them for the quality control, the routine analysis and the dissolution test of the marketing tablets containing ZID and LAM drugs. PMID:26085428

  6. Comparison of a quantitative PCR assay with peripheral blood smear examination for detection and quantitation of Babesia microti infection in humans.

    PubMed

    Wang, Guiqing; Villafuerte, Patrick; Zhuge, Jian; Visintainer, Paul; Wormser, Gary P

    2015-06-01

    Using a real-time quantitative PCR (qPCR), we determined the number of DNA copies/mL of blood of a Babesia microti gene in infected patients. Thirty-six patients (whose median age was 62.5years and 75.0% were male) with at least 1 qPCR-positive blood sample were included in this analysis, including 16 with serial blood samples. Based on testing of serial blood samples, it could be demonstrated that the smear became negative while the qPCR remained positive. A moderate to strong correlation was found between the DNA copy number and the number of infected erythrocytes per milliliter of blood (Pearson's r=0.68, P<0.001). Based on limited data, the DNA copy number fell by a mean of 4.1-12.9% per day on active treatment and by 3.5-7.1% per day off therapy. qPCR methodology may permit systematic evaluations of the relative efficacy of various antiparasitic drug regimens and other therapeutic modalities, although a limitation of such testing is that DNA detection per se does not establish the presence of viable parasites. PMID:25861873

  7. Measurement of lentiviral vector titre and copy number by cross-species duplex quantitative PCR.

    PubMed

    Christodoulou, I; Patsali, P; Stephanou, C; Antoniou, M; Kleanthous, M; Lederer, C W

    2016-01-01

    Lentiviruses are the vectors of choice for many preclinical studies and clinical applications of gene therapy. Accurate measurement of biological vector titre before treatment is a prerequisite for vector dosing, and the calculation of vector integration sites per cell after treatment is as critical to the characterisation of modified cell products as it is to long-term follow-up and the assessment of risk and therapeutic efficiency in patients. These analyses are typically based on quantitative real-time PCR (qPCR), but as yet compromise accuracy and comparability between laboratories and experimental systems, the former by using separate simplex reactions for the detection of endogene and lentiviral sequences and the latter by designing different PCR assays for analyses in human cells and animal disease models. In this study, we validate in human and murine cells a qPCR system for the single-tube assessment of lentiviral vector copy numbers that is suitable for analyses in at least 33 different mammalian species, including human and other primates, mouse, pig, cat and domestic ruminants. The established assay combines the accuracy of single-tube quantitation by duplex qPCR with the convenience of one-off assay optimisation for cross-species analyses and with the direct comparability of lentiviral transduction efficiencies in different species. PMID:26202078

  8. Human Papillomavirus Load Measured by Linear Array Correlates with Quantitative PCR in Cervical Cytology Specimens

    PubMed Central

    Gravitt, Patti E.; Long, Rodney; Schiffman, Mark; Dunn, S. Terence; Carreon, J. Daniel; Allen, Richard A.; Gunja, Munira; Zuna, Rosemary E.; Sherman, Mark E.; Gold, Michael A.; Walker, Joan L.; Wang, Sophia S.

    2012-01-01

    Carcinogenic human papillomavirus (HPV) infections are necessary causes of most anogenital cancers. Viral load has been proposed as a marker for progression to cancer precursors but has been confirmed only for HPV16. Challenges in studying viral load are related to the lack of validated assays for a large number of genotypes. We compared viral load measured by Linear Array (LA) HPV genotyping with the gold standard, quantitative PCR (Q-PCR). LA genotyping and Q-PCR were performed in 143 cytology specimens from women referred to colposcopy. LA signal strength was measured by densitometry. Correlation coefficients and receiver operating characteristic (ROC) analyses were used to evaluate analytical and clinical performance. We observed a moderate to strong correlation between the two quantitative viral load measurements, ranging from an R value of 0.61 for HPV31 to an R value of 0.86 for HPV52. We also observed agreement between visual LA signal strength evaluation and Q-PCR. Both quantifications agreed on the disease stages with highest viral load, which varied by type (cervical intraepithelial neoplasia grade 2 [CIN2] for HPV52, CIN3 for HPV16 and HPV33, and cancer for HPV18 and HPV31). The area under the curve (AUC) for HPV16 Q-PCR at the CIN3 cutoff was 0.72 (P = 0.004), and the AUC for HPV18 LA at the CIN2 cutoff was 0.78 (P = 0.04). Quantification of LA signals correlates with the current gold standard for viral load, Q-PCR. Analyses of viral load need to address multiple infections and type attribution to evaluate whether viral load has clinical value beyond the established HPV16 finding. Our findings support conducting comprehensive studies of viral load and cervical cancer precursors using quantitative LA genotyping data. PMID:22337992

  9. Quantitative Real-Time PCR Assay for QPX (Thraustochytriidae), a Parasite of the Hard Clam (Mercenaria mercenaria)▿

    PubMed Central

    Liu, Qianqian; Allam, Bassem; Collier, Jackie L.

    2009-01-01

    We developed a real-time quantitative PCR (qPCR) assay targeting the rRNA internal transcribed spacer region of the hard clam pathogen QPX. The qPCR assay was more sensitive than was histology in detecting clams with light QPX infections. QPX was detected in 4 of 43 sediment samples but in none of 40 seawater samples. PMID:19465523

  10. Reference gene selection for gene expression studies in lily using quantitative real-time PCR.

    PubMed

    Zhang, M F; Liu, Q; Jia, G X

    2016-01-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is an important technology used to analyze gene-expression levels. Reference genes, which are assumed to be expressed consistently across various developmental stages and in different tissues, were selected for expression level analysis. Using digital gene expression technology, we selected nine reference genes (18S, EF, CYCOL, SAND, GAPDH, ACTIN, BHLH, TIP, and Clathrin) as candidate reference genes for further study. Using three different analysis methods (GeNorm, NormFinder, and BestKeeper), a total of 144 lily (Lilium x formolongi "Raizan 3") samples were analyzed. The samples were collected from four different tissues under various developmental stages. In addition, leaves treated with different plant hormones were collected and analyzed. The data showed that the stability of the nine reference genes differed among samples, but TIP, EF, Clathrin, and BHLH could be identified as the most stable genes overall. In addition, the relative expression level of LfFT in different lily tissues with the competence to flower was also analyzed to verify the selected reference genes. This study constitutes an important source for selecting reference genes when analyzing the expression patterns of flowering time and floral development regulation genes in lily cultivars. PMID:27173307

  11. Enhanced detection of surface-associated bacteria in indoor environments by quantitative PCR.

    PubMed

    Buttner, M P; Cruz-Perez, P; Stetzenbach, L D

    2001-06-01

    Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling

  12. Quantitative RT-PCR assessment of melanoma cells in peripheral blood during immunotherapy for metastatic melanoma.

    PubMed

    Schmidt, H; Sørensen, B S; von der Maase, H; Bang, C; Agger, R; Hokland, M; Nexo, E

    2002-12-01

    Circulating malignant cells in peripheral blood are thought to be precursors and surrogate markers of distant metastases and hence markers of a poor clinical outcome. In this study, we used the detection of MART-1 and tyrosinase (TYR) mRNA with a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to identify circulating melanoma cells. Blood samples were obtained from 35 patients with metastatic melanoma before, during and after treatment with interleukin-2, interferon-alpha and cisplatin. In addition, MART-1 and TYR protein was identified by immunohistochemistry in consecutive biopsies from 15 of the patients. Analysis of three daily blood samples for 3 days demonstrated that four out of 11 patients examined were negative for both markers on all occasions, and two patients were positive for both markers on all occasions but one. The remaining five patients showed sporadic low positive results for one or the other of the two markers. By comparing the immunohistochemistry results from consecutive biopsies with the RT-PCR results, we demonstrated that patients with MART-1 and TYR protein in their tumour cells had circulating MART-1 and TYR mRNA in 77% and 54% of the cases, respectively. During treatment, the majority of patients who were positive for MART-1 and TYR mRNA converted to being negative. However, these conversions did not significantly correlate with objective response. The presence of TYR mRNA in one of the first two samples showed a trend towards being an independent prognostic factor for poor survival. PMID:12459648

  13. Development of TaqMan-Based Quantitative PCR for Sensitive and Selective Detection of Toxigenic Clostridium difficile in Human Stools

    PubMed Central

    Kubota, Hiroyuki; Sakai, Takafumi; Gawad, Agata; Makino, Hiroshi; Akiyama, Takuya; Ishikawa, Eiji; Oishi, Kenji

    2014-01-01

    Background Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation. Methods TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC). Results The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA−TcdB+, and TcdA−TcdB− types), TcdB-producing strains (TcdA+TcdB+ and TcdA−TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA−TcdB− strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota. Conclusion Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of

  14. Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

    PubMed Central

    Katayama, Hiroyuki; Furumai, Hiroaki

    2014-01-01

    Reverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log10-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances. PMID:25527552

  15. Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR.

    PubMed

    Henrich, Timothy J; Gallien, Sebastien; Li, Jonathan Z; Pereyra, Florencia; Kuritzkes, Daniel R

    2012-12-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  16. Low-Level Detection and Quantitation of Cellular HIV-1 DNA and 2-LTR Circles Using Droplet Digital PCR

    PubMed Central

    Henrich, Timothy J.; Gallien, Sebastien; Li, Jonathan Z.; Pereyra, Florencia; Kuritzkes, Daniel R.

    2012-01-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia was also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  17. Detecting Polychlorinated Biphenyls by Ah Receptor and Fluorescence Quantitative PCR with Exonuclease

    NASA Astrophysics Data System (ADS)

    Zhao, Xiaoxiang; Zhuang, Huisheng

    2010-11-01

    Tetrachlorobiphenyls as ligands were cultivated with goldfish, Ah receptors were extracted from the liver of goldfish and purified by hydroxyapatite. The complex of TCB ligands-receptors were analyzed by Surface Plasmon Resonance. DNA probes were amplified by PCR using Primers F1 and F2 with the DNA recognition site of responsive enhancer. DNA probes bound to the complex were not digested by exonuclease. The DNA that bound to the complex was quantified by real time PCR. A standard curve with TCB concentration to Ct values was obtained in the range of 10-12mol/L to 10-8 mol/L, according to TCB concentration in samples. The detection limit of the assay was below 10-12mol/L of TCB. Compared with HPLC, this assay is much more sensitive. These results suggest that fluorescence quantitative PCR with exonuclease by Ah receptors fits for detection of trace PCB.

  18. Enteroaggregative Escherichia coli quantification in children stool samples using quantitative PCR

    PubMed Central

    LIMA, ILA FERNANDA NUNES; QUETZ, JOSIANE DA SILVA; GUERRANT, RICHARD LITTLETON; NATARO, JAMES P.; HOUPT, ERIC R.; LIMA, ALDO ANGELO MOREIRA; HAVT, ALEXANDRE

    2012-01-01

    Enteroaggregative Escherichia coli (EAEC) is a common cause of infectious diarrhea, especially in children living in poor-resource countries. In this article, we present a SYBR Green-based real-time polymerase chain reaction (qPCR) method for quantitative detection of EAEC in DNA directly extracted from human stool samples. In order to test the proposed qPCR system, we examined specificity, sensitivity, repeatability, and also the degree of DNA extraction efficiency using EAEC strain 042 spiked into EAEC-free stool sample. The specificity of this assay was proved using 6 strains of EAEC, 7 strains of other E. coli types, and one strain of Shigella. The detection limit of qPCR was 67 CFU/reaction. In naturally infected stool samples, we found EAEC in quantities varying from 6.7 × 105 to 2 × 109 CFU/g of feces. We could not detect any reduction after stool DNA extraction for the amounts of 107 and 106 CFU/mL of spiked EAEC. This qPCR assay is simple, rapid, reproducible, sensitive, specific, and allows rapid EAEC quantification to be used in a variety of further EAEC studies. This new quantitative method provides a relatively simple means to quantifify EAEC, which will likely be key to understanding the pathophysiology and impact of EAEC infection. PMID:23216208

  19. Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

    PubMed Central

    Ludlow, Andrew T.; Robin, Jerome D.; Sayed, Mohammed; Litterst, Claudia M.; Shelton, Dawne N.; Shay, Jerry W.; Wright, Woodring E.

    2014-01-01

    The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase. PMID:24861623

  20. Quantitative PCR for Tracking the Megaplasmid-Borne Biodegradation Potential of a Model Sphingomonad

    PubMed Central

    Hartmann, Erica M.; Badalamenti, Jonathan P.; Krajmalnik-Brown, Rosa

    2012-01-01

    We developed a quantitative PCR method for tracking the dxnA1 gene, the initial, megaplasmid-borne gene in Sphingomonas wittichii RW1's dibenzo-p-dioxin degradation pathway. We used this method on complex environmental samples and report on growth of S. wittichii RW1 in landfill leachate, thus furnishing a novel tool for monitoring megaplasmid-borne, dioxygenase-encoding genes. PMID:22492441

  1. Real-time PCR-based assay for quantitative detection of Hematodinium sp. in the blue crab Callinectes sapidus.

    PubMed

    Nagle, L; Place, A R; Schott, E J; Jagus, R; Messick, G; Pitula, J S

    2009-03-01

    Hematodinium sp. is a parasitic dinoflagellate infecting the blue crab Callinectes sapidus and other crustaceans. PCR-based assays are currently being used to identify infections in crabs that would have been undetectable by traditional microscopic examination. We therefore sought to define the limits of quantitative PCR (qPCR) detection within the context of field collection protocols. We present a qPCR assay based on the Hematodinium sp. 18S rRNA gene that can detect 10 copies of the gene per reaction. Analysis of a cell dilution series vs. defined numbers of a cloned Hematodinium sp. 18S rRNA gene suggests a copy number of 10,000 per parasite and predicts a sensitivity of 0.001 cell equivalents. In practice, the assays are based on analysis of 1% of the DNA extracted from 200 microl of serum, yielding a theoretical detection limit of 5 cells ml(-1) hemolymph, assuming that 1 cell is present per sample. When applied to a limited field survey of blue crabs collected in Maryland coastal bays from May to August 2005, 24 of 128 crabs (18.8%) were identified as positive for Hematodinium sp. infection using qPCR. In comparison, only 6 of 128 crabs (4.7%) were identified as positive using traditional hemolymph microscopic examination. The qPCR method also detected the parasite in gill, muscle, heart and hepatopancreas tissues, with 17.2% of the crabs showing infection in at least one of these tissues. Importantly, it is now possible to enumerate parasites within defined quantities of crab tissue, which permits collection of more detailed information on the epizootiology of the pathogen. PMID:19419009

  2. PCR Diagnosis of Pneumocystis Pneumonia: a Bivariate Meta-Analysis

    PubMed Central

    Lu, Yuan; Ling, Guoya; Qiang, Chenyi; Ming, Qinshou; Wu, Cong; Wang, Ke; Ying, Zouxiao

    2011-01-01

    We undertook a bivariate meta-analysis to assess the overall accuracy of respiratory specimen PCR assays for diagnosing Pneumocystis pneumonia. The summary sensitivity and specificity were 0.99 (95% confidence interval, 0.96 to 1.00) and 0.90 (0.87 to 0.93). Subgroup analyses showed that quantitative PCR analysis and the major surface glycoprotein gene target had the highest specificity value (0.93). Respiratory specimen PCR results are sufficient to confirm or exclude the disease for at-risk patients suspected of having Pneumocystis pneumonia. PMID:22012008

  3. [Development and validation of event-specific quantitative PCR method for genetically modified maize LY038].

    PubMed

    Mano, Junichi; Masubuchi, Tomoko; Hatano, Shuko; Futo, Satoshi; Koiwa, Tomohiro; Minegishi, Yasutaka; Noguchi, Akio; Kondo, Kazunari; Akiyama, Hiroshi; Teshima, Reiko; Kurashima, Takeyo; Takabatake, Reona; Kitta, Kazumi

    2013-01-01

    In this article, we report a novel real-time PCR-based analytical method for quantitation of the GM maize event LY038. We designed LY038-specific and maize endogenous reference DNA-specific PCR amplifications. After confirming the specificity and linearity of the LY038-specific PCR amplification, we determined the conversion factor required to calculate the weight-based content of GM organism (GMO) in a multilaboratory evaluation. Finally, in order to validate the developed method, an interlaboratory collaborative trial according to the internationally harmonized guidelines was performed with blind DNA samples containing LY038 at the mixing levels of 0, 0.5, 1.0, 5.0 and 10.0%. The precision of the method was evaluated as the RSD of reproducibility (RSDR), and the values obtained were all less than 25%. The limit of quantitation of the method was judged to be 0.5% based on the definition of ISO 24276 guideline. The results from the collaborative trial suggested that the developed quantitative method would be suitable for practical testing of LY038 maize. PMID:23470871

  4. Detection of Legionella species in environmental water by the quantitative PCR method in combination with ethidium monoazide treatment.

    PubMed

    Inoue, Hiroaki; Takama, Tomoko; Yoshizaki, Miwa; Agata, Kunio

    2015-01-01

    We detected Legionella species in 111 bath water samples and 95 cooling tower water samples by using a combination of conventional plate culture, quantitative polymerase chain reaction (qPCR) and qPCR combined with ethidium monoazide treatment (EMA-qPCR) methods. In the case of bath water samples, Legionella spp. were detected in 30 samples by plate culture, in 85 samples by qPCR, and in 49 samples by EMA-qPCR. Of 81 samples determined to be Legionella-negative by plate culture, 56 and 23 samples were positive by qPCR and EMA-qPCR, respectively. Therefore, EMA treatment decreased the number of Legionella-positive bath water samples detected by qPCR. In contrast, EMA treatment had no effect on cooling tower water samples. We therefore expect that EMA-qPCR is a useful method for the rapid detection of viable Legionella spp. from bath water samples. PMID:25817816

  5. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay

    PubMed Central

    Shen, Shu-Min; Chou, Ming-Yuan; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

    2015-01-01

    Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 102 and 3.3 × 105 cells/l in river water and 72.1–5.7 × 106 cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors. PMID:26184706

  6. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    PubMed

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  7. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping

    PubMed Central

    Lee, Han B.; Schwab, Tanya L.; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L.; Cervera, Roberto Lopez; McNulty, Melissa S.; Bostwick, Hannah S.; Clark, Karl J.

    2016-01-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  8. Multi-target PCR analysis by capillary electrophoresis and laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Lu, Wei; Han, Dai-Shu; Yuan, Ju; Andrieu, Jean-Marie

    1994-03-01

    Quantitative analysis of polymerase chain reaction (PCR) amplified HIV-1 DNA or cDNA fragments is attained using an automated system that combines capillary-gel electrophoresis (CGE) for high-efficiency separation and laser-induced fluorescence (LIF) for high-sensitivity detection. This system enables the detection of PCR-amplified multiple target DNA or cDNA in the same tube by a single injection with high precision.

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

    PubMed Central

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

    2015-01-01

    Background 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. Results 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

  10. Detection of a Molecular Biomarker for Zygomycetes by Quantitative PCR Assays of Plasma, Bronchoalveolar Lavage, and Lung Tissue in a Rabbit Model of Experimental Pulmonary Zygomycosis▿

    PubMed Central

    Kasai, Miki; Harrington, Susan M.; Francesconi, Andrea; Petraitis, Vidmantas; Petraitiene, Ruta; Beveridge, Mara G.; Knudsen, Tena; Milanovich, Jeffery; Cotton, Margaret P.; Hughes, Johanna; Schaufele, Robert L.; Sein, Tin; Bacher, John; Murray, Patrick R.; Kontoyiannis, Dimitrios P.; Walsh, Thomas J.

    2008-01-01

    We developed two real-time quantitative PCR (qPCR) assays, targeting the 28S rRNA gene, for the diagnosis of zygomycosis caused by the most common, clinically significant Zygomycetes. The amplicons of the first qPCR assay (qPCR-1) from Rhizopus, Mucor, and Rhizomucor species were distinguished through melt curve analysis. The second qPCR assay (qPCR-2) detected Cunninghamella species using a different primer/probe set. For both assays, the analytic sensitivity for the detection of hyphal elements from germinating sporangiospores in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates from rabbits was 1 to 10 sporangiospores/ml. Four unique and clinically applicable models of invasive pulmonary zygomycosis served as surrogates of human infections, facilitating the validation of these assays for potential diagnostic utility. For qPCR-1, 5 of 98 infarcted lung specimens were positive by qPCR and negative by quantitative culture (qCx). None were qCx positive only. Among 23 BAL fluid samples, all were positive by qPCR, while 22 were positive by qCx. qPCR-1 detected Rhizopus and Mucor DNA in 20 (39%) of 51 serial plasma samples as early as day 1 postinoculation. Similar properties were observed for qPCR-2, which showed greater sensitivity than qCx for BAL fluid (100% versus 67%; P = 0.04; n = 15). The assay detected Cunninghamella DNA in 18 (58%) of 31 serial plasma samples as early as day 1 postinoculation. These qPCR assays are sensitive and specific for the detection of Rhizopus, Mucor, Rhizomucor, and Cunninghamella species and can be used for the study and detection of infections caused by these life-threatening pathogens. PMID:18845827