Artículos de revistas: "PCR real time quantitativa"

1

Heid, C. A., J. Stevens, K. J. Livak y P. M. Williams. "Real time quantitative PCR." Genome Research 6, n.º 10 (1 de octubre de 1996): 986–94. http://dx.doi.org/10.1101/gr.6.10.986.

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2

Schmittgen, Thomas D. "Real-Time Quantitative PCR". Methods 25, n.º 4 (diciembre de 2001): 383–85. http://dx.doi.org/10.1006/meth.2001.1260.

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3

RAZA, ABIDA y NAUREEN A KHATTAK. "REAL TIME PCR;". Professional Medical Journal 19, n.º 06 (3 de noviembre de 2012): 751–59. http://dx.doi.org/10.29309/tpmj/2012.19.06.2455.

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In recent years, real-time PCR has come forward as a robust and widely used molecular technique in clinical and biologicalsettings. Although it can detect very minute quantities of target nucleic acid, but quantification of specific nucleic acids is not an easy task.Accurate and precise quantification is hampered by a number of factors that may include assay development and validation, fluorophoresselection, handling during sample preparation, storage, reaction procedures, and batch analysis conditions. Even minor variations aresignificantly magnified by the exponential nature of this technique. Current review gives an insight of the advantages, limitations, assaychemistries, quantitation parameters, and quality control issues related to this technology. Moreover it will also highlight the utilization of Realtime PCR in clinical oncology, virology, microbiology, and gene expression studies.

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4

Choi, Yeon-Jae, Sun-Ho Kim, Min-Jeong Gu, Han-Na Choe, Dong-Un Kim, Sang-Bum Cho, Su-Ki Kim, Che-Ok Jeon, Gui-Seok Bae y Sang-Seok Lee. "Quantitative Real-time PCR using Lactobacilli as Livestock Probiotics". Journal of Life Science 20, n.º 12 (30 de diciembre de 2010): 1896–901. http://dx.doi.org/10.5352/jls.2010.20.12.1896.

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5

Bell, Andrew S. y Lisa C. Ranford-Cartwright. "Real-time quantitative PCR in parasitology". Trends in Parasitology 18, n.º 8 (agosto de 2002): 338–42. http://dx.doi.org/10.1016/s1471-4922(02)02331-0.

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6

Wong, Marisa L. y Juan F. Medrano. "Real-time PCR for mRNA quantitation". BioTechniques 39, n.º 1 (julio de 2005): 75–85. http://dx.doi.org/10.2144/05391rv01.

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7

Bustin, S. A., V. Benes, T. Nolan y M. W. Pfaffl. "Quantitative real-time RT-PCR – a perspective". Journal of Molecular Endocrinology 34, n.º 3 (junio de 2005): 597–601. http://dx.doi.org/10.1677/jme.1.01755.

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The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.

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8

Sochivko, D. G., A. A. Fedorov, D. A. Varlamov, V. E. Kurochkin y R. V. Petrov. "Accuracy of quantitative real-time PCR analysis". Doklady Biochemistry and Biophysics 449, n.º 1 (marzo de 2013): 105–8. http://dx.doi.org/10.1134/s1607672913020154.

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9

Arya, Manit, Iqbal S. Shergill, Magali Williamson, Lyndon Gommersall, Neehar Arya y Hitendra RH Patel. "Basic principles of real-time quantitative PCR". Expert Review of Molecular Diagnostics 5, n.º 2 (marzo de 2005): 209–19. http://dx.doi.org/10.1586/14737159.5.2.209.

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10

Boulay, J. L., J. Reuter, R. Ritschard, L. Terracciano, R. Herrmann y C. Rochlitz. "Gene Dosage by Quantitative Real-Time PCR". BioTechniques 27, n.º 2 (agosto de 1999): 228–32. http://dx.doi.org/10.2144/99272bm03.

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11

Gil, Marcel E. y Thérèsa L. Coetzer. "Real-Time Quantitative PCR of Telomere Length". Molecular Biotechnology 27, n.º 2 (2004): 169–72. http://dx.doi.org/10.1385/mb:27:2:169.

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12

Ginzinger, David G. "Gene quantification using real-time quantitative PCR". Experimental Hematology 30, n.º 6 (junio de 2002): 503–12. http://dx.doi.org/10.1016/s0301-472x(02)00806-8.

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13

Holzapfel, Bianca y Lucia Wickert. "Die quantitative Real-Time-PCR (qRT-PCR). Methoden und Anwendungsgebiete". Biologie in unserer Zeit 37, n.º 2 (abril de 2007): 120–26. http://dx.doi.org/10.1002/biuz.200610332.

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14

Jeong, Hye Mi y Kwang Yup Kim. "Quantitative Analysis of Feline Calicivirus Inactivation using Real-time RT-PCR". Journal of Food Hygiene and Safety 29, n.º 1 (30 de marzo de 2014): 31–39. http://dx.doi.org/10.13103/jfhs.2014.29.1.031.

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15

Lee, Jae Il y In Seop Kim. "TaqMan Probe Real-Time PCR for Quantitative Detection of Mycoplasma during Manufacture of Biologics". KSBB Journal 29, n.º 5 (30 de octubre de 2014): 361–71. http://dx.doi.org/10.7841/ksbbj.2014.29.5.361.

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16

Heo, Jeong, Won Ook Go, Gwang Ha Kim, Dae Hwan Kang, Geun Am Song, Mong Cho, Hyung Hoi Kim y Eeu Yup Lee. "HBV DNA Quantitation Using Real-time PCR". Annals of Laboratory Medicine 26, n.º 6 (1 de diciembre de 2006): 424–30. http://dx.doi.org/10.3343/kjlm.2006.26.6.424.

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17

Mullah, Bashar, Paul Wyatt, Junko Stevens, Alex Andrus y Kenneth J. Livak. "Automated real-time PCR detection and quantitation". Collection of Czechoslovak Chemical Communications 61, s1 (1996): 287–89. http://dx.doi.org/10.1135/cccc1996s287.

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18

White, Jonathan R. "Gene expression quantitation by real-time PCR". BJU International 111, n.º 1 (20 de diciembre de 2012): 157–58. http://dx.doi.org/10.1111/j.1464-410x.2012.11706.x.

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19

Choe, Myeong-Eun, In-Jung Lee y Jae-Ho Shin. "Assessment of Korean Paddy Soil Microbial Community Structure by Use of Quantitative Real-time PCR Assays". Korean Journal of Environmental Agriculture 30, n.º 4 (31 de diciembre de 2011): 367–76. http://dx.doi.org/10.5338/kjea.2011.30.4.367.

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20

Kosinova, E., I. Psikal, B. Robesova y K. Kovarcik. "Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry". Veterinární Medicína 52, No. 6 (7 de enero de 2008): 253–61. http://dx.doi.org/10.17221/1882-vetmed.

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Quantitative real-time RT-PCR (qRT-PCR) assay was developed for the detection and quantification of bovine viral diarrhea virus (BVDV) in clinical samples from persistently infected cattle. qRT-PCR was optimized to quantify the number of BVD virus copies using Light Cycler<sup>®</sup> detection system and intercalation fluorogenic dye SYBR Green I. A universal set of primers was selected from a highly conserved 5′ untranslated region (5′UTR) to detect BVDV type I and II simultaneously. Quantification of BVDV cDNA was accomplished using a calibration curve generated from 10-fold serial dilutions of standard plasmid DNA in the range 1−10<sup>8</sup> copies/μl. Analysis of 290 bp amplicons enabled monitoring of the viral RNA/BVDV level in a total of five BVDV strains (BVD-NADL, A03/3004, DB03/2943, KA04/3124, KV05/3412) and sixteen bulk milk samples, and in bovine sera of persistent carriers originating from Czech farms, as well as in a batch of calf serum for cell culture. Melting temperatures of amplicons (Tm) of BVDV strains of the same genotype group I as the NADL reference strain showed variability of the thermal points, however significant differences were observed in Tm values between the representatives of genotype group I and II. Low concentrations of BVD virus in bulk milk samples were also qualitatively identified by conventional RT-PCR. Highly reproducible data were obtained as the coefficients of variation of threshold cycles values in intra-assay and inter-assay were less than 0.85% and 2.76%, respectively. The results give enough evidence of suitability of qRT-PCR assay for quantitative analysis of BVDV in clinical samples.

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21

Bastien, P., G. W. Procop y U. Reischl. "Quantitative Real-Time PCR Is Not More Sensitive than "Conventional" PCR". Journal of Clinical Microbiology 46, n.º 6 (9 de abril de 2008): 1897–900. http://dx.doi.org/10.1128/jcm.02258-07.

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22

Skarratt, Kristen K. y Stephen J. Fuller. "Quantitative real-time PCR eliminates false-positives in colony screening PCR". Journal of Microbiological Methods 96 (enero de 2014): 99–100. http://dx.doi.org/10.1016/j.mimet.2013.11.011.

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23

Si, Chen, Huang Kun-Lun, Xu Wen-Tao, Li Yuan y Luo Yun-Bo. "Real-time quantitative PCR detection ofEscherichia coliO157:H7". Chinese Journal of Agricultural Biotechnology 4, n.º 1 (abril de 2007): 15–19. http://dx.doi.org/10.1017/s1479236207001349.

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AbstractA rapid and accurate real-time quantitative polymerase chain reaction (real-time PCR) method with SYBR Green I was established for detectingEscherichia coliO157:H7. A pair of primers were designed to amplify theeaegene. The dissociation curves showed that the amplification product was very specific. The optimal conditions and standard curve were established. The result indicated that real-time PCR was 1000 times more sensitive than ordinary PCR.

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24

Fossey, Sallyanne C., Andrea Ferreira-Gonzalez, Carleton T. Garrett, Catherine I. Dumur y Cindy L. Vnencak-Jones. "BCRABL Transcript Detection by Quantitative Real-Time PCR". Molecular Diagnosis 9, n.º 4 (2005): 187–93. http://dx.doi.org/10.2165/00066982-200509040-00004.

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25

Shively, L., L. Chang, J. M. LeBon, Q. Liu, A. D. Riggs y J. Singer-Sam. "Real-Time PCR Assay for Quantitative Mismatch Detection". BioTechniques 34, n.º 3 (marzo de 2003): 498–504. http://dx.doi.org/10.2144/03343st01.

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26

Lin, Mei-Hui, Tse-Ching Chen, Tseng-tong Kuo, Ching-Chung Tseng y Ching-Ping Tseng. "Real-Time PCR for Quantitative Detection ofToxoplasma gondii". Journal of Clinical Microbiology 38, n.º 11 (2000): 4121–25. http://dx.doi.org/10.1128/jcm.38.11.4121-4125.2000.

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The protozoan Toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of T. gondii. Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the T. gondii B1 gene. After 40 PCR cycles, the cycle threshold values (CT) indicative of the quantity of the target gene were determined. Typically, a CT of 25.09 was obtained with DNA from 500 tachyzoites of the T. gondii RH strain. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hence, we were able to quantitatively detect as little as 0.05 T. gondii tachyzoite in an assay. When tested with 30 paraffin-embedded fetal tissue sections, 10 sections (33%) showed a CT of <40 and were scored as positive for this test. These results were consistent with those obtained through our nested-PCR control experiments. We have developed a rapid, sensitive, and quantitative real-time PCR for detection of T. gondii. The advantages of this technique for the diagnosis of toxoplasmosis in a clinical laboratory are discussed.

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27

Sanburn, N. y K. Cornetta. "Rapid titer determination using quantitative real-time PCR". Gene Therapy 6, n.º 7 (julio de 1999): 1340–45. http://dx.doi.org/10.1038/sj.gt.3300948.

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28

Rintala, Helena y Aino Nevalainen. "Quantitative measurement of streptomycetes using real-time PCR". Journal of Environmental Monitoring 8, n.º 7 (2006): 745. http://dx.doi.org/10.1039/b602485h.

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29

Nonnenmacher, Claudia, Alexander Dalpke, Reinier Mutters y Klaus Heeg. "Quantitative detection of periodontopathogens by real-time PCR". Journal of Microbiological Methods 59, n.º 1 (octubre de 2004): 117–25. http://dx.doi.org/10.1016/j.mimet.2004.06.006.

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30

Love, John L., Paula Scholes, Brent Gilpin, Marion Savill, Susan Lin y Laly Samuel. "Evaluation of uncertainty in quantitative real-time PCR". Journal of Microbiological Methods 67, n.º 2 (noviembre de 2006): 349–56. http://dx.doi.org/10.1016/j.mimet.2006.04.005.

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31

Sawyer, Jason, Clare Wood, Della Shanahan, Sally Gout y David McDowell. "Real-time PCR for quantitative meat species testing". Food Control 14, n.º 8 (diciembre de 2003): 579–83. http://dx.doi.org/10.1016/s0956-7135(02)00148-2.

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32

Väänänen, Riina-Minna, Maria Rissanen, Otto Kauko, Siina Junnila, Ville Väisänen, Jussi Nurmi, Kalle Alanen, Martti Nurmi y Kim Pettersson. "Quantitative real-time RT-PCR assay for PCA3". Clinical Biochemistry 41, n.º 1-2 (enero de 2008): 103–8. http://dx.doi.org/10.1016/j.clinbiochem.2007.10.009.

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33

Fossey, Sallyanne C., Andrea Ferreira-Gonzalez, Carleton T. Garrett, Catherine I. Dumur y Cindy L. Vnencak-Jones. "BCRABL Transcript Detection by Quantitative Real-Time PCR". Molecular Diagnosis 9, n.º 4 (diciembre de 2005): 187–93. http://dx.doi.org/10.1007/bf03260090.

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34

Ahmad, Ashraf I. y Jahan B. Ghasemi. "New FRET primers for quantitative real-time PCR". Analytical and Bioanalytical Chemistry 387, n.º 8 (17 de febrero de 2007): 2737–43. http://dx.doi.org/10.1007/s00216-007-1123-4.

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35

Bookout, Angie L., Carolyn L. Cummins, David J. Mangelsdorf, Jean M. Pesola y Martha F. Kramer. "High-Throughput Real-Time Quantitative Reverse Transcription PCR". Current Protocols in Molecular Biology 73, n.º 1 (enero de 2006): 15.8.1–15.8.28. http://dx.doi.org/10.1002/0471142727.mb1508s73.

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36

Morpeth, Susan C., Jim F. Huggett, David R. Murdoch y J. Anthony G. Scott. "Making standards for quantitative real-time pneumococcal PCR". Biomolecular Detection and Quantification 2 (diciembre de 2014): 1–3. http://dx.doi.org/10.1016/j.bdq.2014.11.003.

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37

Roque-Biewer, Mimi, Mark Shannon, Kathryn Hunkapiller, Alexandra Fuller, Jeff Bluestone, Vicki Seyfert-Margolis y David Ruff. "Applications and advances in real-time quantitative PCR". Human Immunology 65, n.º 9-10 (septiembre de 2004): S118. http://dx.doi.org/10.1016/j.humimm.2004.07.225.

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38

Whittle, Martin R. y Denilce R. Sumita. "Quadruplex real-time PCR for forensic DNA quantitation". Forensic Science International: Genetics Supplement Series 1, n.º 1 (agosto de 2008): 86–88. http://dx.doi.org/10.1016/j.fsigss.2007.08.012.

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39

Hodek, J., J. Ovesná y L. Kučera. "Interferences of PCR effectivity: importance for quantitative analyse". Czech Journal of Food Sciences 27, Special Issue 2 (3 de enero de 2010): 42–49. http://dx.doi.org/10.17221/677-cjfs.

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Importance of the Polymerase chain reaction (PCR) have already crossed the border of mere target DNA sequence present or absence analysis. For number analyses e.g. Genetically Modified Organisms (GMOs) or gene expression assesment the DNA quantification is demanded. Real-time (or quantitative) PCR is the most used tool for nucleic acids quantification. PCR efficiency has relevant importance on DNA quantification – it should be almost same for each PCR and its value should varied between 90–100%. There are a lot of PCR enhancers and inhibitors well known. We described impact of used DNA solvent and used laboratory plastic on real-time PCR efficiency.

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40

Jones, Gerwyn M., Eloise Busby, Jeremy A. Garson, Paul R. Grant, Eleni Nastouli, Alison S. Devonshire y Alexandra S. Whale. "Digital PCR dynamic range is approaching that of real-time quantitative PCR". Biomolecular Detection and Quantification 10 (diciembre de 2016): 31–33. http://dx.doi.org/10.1016/j.bdq.2016.10.001.

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41

Stein, Jason B., Malcolm McGinnis, Ian McLaughlin, Pete Krausa, Steve Beckert y Kathy Lazaruk. "118-P: Real-time quantitative PCR vs. STR PCR for chimerism analysis". Human Immunology 69 (octubre de 2008): S66. http://dx.doi.org/10.1016/j.humimm.2008.08.137.

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42

Oriero, C. E., D. Nwakanma, S. Sesay y D. Conway. "Determination of Parasite Clearance Time in Antimalarial Drug Trials Using Real-Time Quantitative PCR (PCR)". International Journal of Infectious Diseases 12 (diciembre de 2008): e311. http://dx.doi.org/10.1016/j.ijid.2008.05.833.

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43

Nitsche, Andreas, Mathias Büttner, Sonja Wilhelm, Georg Pauli y Hermann Meyer. "Real-Time PCR Detection of Parapoxvirus DNA",. Clinical Chemistry 52, n.º 2 (1 de febrero de 2006): 316–19. http://dx.doi.org/10.1373/clinchem.2005.060335.

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Abstract Background: Detection of parapoxviruses is important in various animals as well as in humans as zoonotic infections. Reliable detection of parapoxviruses is fundamental for the exclusion of other rash-causing illnesses, for both veterinarians and medical practitioners. To date, however, no real-time PCR assay for the detection of parapoxviruses has been reported. Methods: A minor groove binder–based quantitative real-time PCR assay targeting the B2L gene of parapoxviruses was developed on the ABI Prism and the LightCycler platforms. Results: The real-time PCR assay successfully amplified DNA fragments from a total of 41 parapoxvirus strains and isolates representing the species orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, and sealpoxvirus. Probit analysis gave a limit of detection of 4.7 copies per assay (95% confidence interval, 3.7–6.8 copies per reaction). Scabs contain a sufficient amount of parapoxvirus DNA and can therefore be used for PCR without any DNA preparation step. No cross-reactivity to human, bovine, or sheep genomic DNA or other DNA viruses, including orthopoxviruses, molluscum contagiosum viruses, and yaba-like disease viruses, was observed. Conclusion: The presented assay is suitable for the detection of parapoxvirus infections in clinical material of human and animal origin.

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44

OKUNISHI, Tomoya, Sumiko NAKAMURA y Ken’ichi OHTSUBO. "Quantitative Identification of Rice Cultivars by Real-Time PCR". Food Science and Technology Research 11, n.º 3 (2005): 344–48. http://dx.doi.org/10.3136/fstr.11.344.

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45

Pahl, Andreas, Uta Kühlbrandt, Kay Brune, Martin Röllinghoff y André Gessner. "Quantitative Detection of Borrelia burgdorferi by Real-Time PCR". Journal of Clinical Microbiology 37, n.º 6 (1999): 1958–63. http://dx.doi.org/10.1128/jcm.37.6.1958-1963.1999.

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Currently, no easy and reliable methods allowing for the quantification of Borrelia burgdorferi in tissues of infected humans or animals are available. Due to the lack of suitable assays to detect B. burgdorferi CFU and the qualitative nature of the currently performed PCR assays, we decided to exploit the recently developed real-time PCR. This technology measures the release of fluorescent oligonucleotides during the PCR. Flagellin of B. burgdorferi was chosen as the target sequence. A linear quantitative detection range of 5 logs with a calculated detection limit of one to three spirochetes per assay reaction mixture was observed. The fact that no signals were obtained with closely related organisms such as Borrelia hermsii argues for a high specificity of this newly developed method. A similar method was developed to quantify mouse actin genomic sequences to allow for the standardization of spirochete load. The specificity and sensitivity of the B. burgdorferi and the actin real-time PCR were not altered when samples were spiked with mouse cells or spirochetes, respectively. To evaluate the applicability of the real-time PCR, we used the mouse model of Lyme disease. The fate of B. burgdorferi was monitored in different tissues from inbred mice and from mice treated with antibiotics. Susceptible C3H/HeJ mice had markedly higher burdens of bacterial DNA than resistant BALB/c mice, and penicillin G treatment significantly reduced the numbers of spirochetes. Since these results show a close correlation between clinical symptoms and bacterial burden of tissues, we are currently analyzing human biopsy specimens to evaluate the real-time PCR in a diagnostic setting.

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46

Locatelli, Giuseppe, Fabio Santoro, Fabrizio Veglia, Alberto Gobbi, Paolo Lusso y Mauro S. Malnati. "Real-Time Quantitative PCR for Human Herpesvirus 6 DNA". Journal of Clinical Microbiology 38, n.º 11 (2000): 4042–48. http://dx.doi.org/10.1128/jcm.38.11.4042-4048.2000.

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The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5′ nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 106 viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys.

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47

Lyons, Sharon R., Ann L. Griffen y Eugene J. Leys. "Quantitative Real-Time PCR forPorphyromonas gingivalis and Total Bacteria". Journal of Clinical Microbiology 38, n.º 6 (2000): 2362–65. http://dx.doi.org/10.1128/jcm.38.6.2362-2365.2000.

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48

He, Q., J. P. Wang, M. Osato y L. B. Lachman. "Real-Time Quantitative PCR for Detection of Helicobacter pylori". Journal of Clinical Microbiology 40, n.º 10 (1 de octubre de 2002): 3720–28. http://dx.doi.org/10.1128/jcm.40.10.3720-3728.2002.

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49

Goh, Jane, Joong W. Lee, Jordan Laser y Silvia Spitzer. "Monitoring Quality Assurance in Quantitative Real-Time PCR Tests". American Journal of Clinical Pathology 138, suppl 2 (1 de noviembre de 2012): A365. http://dx.doi.org/10.1093/ajcp/138.suppl2.174.

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50

MELIANI, LEILA, MICHEL DEVELOUX, MARIE MARTEAU-MILTGEN, DENIS MAGNE, VERONIQUE BARBU, JEAN-LOUIS POIROT y PATRICIA ROUX. "Real Time Quantitative PCR Assay for Pneumocystis jirovecii Detection". Journal of Eukaryotic Microbiology 50, s1 (julio de 2003): 651. http://dx.doi.org/10.1111/j.1550-7408.2003.tb00670.x.

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Artículos de revistas sobre el tema "PCR real time quantitativa"

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