Academic literature on the topic 'Real-Time PCR technique'
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Journal articles on the topic "Real-Time PCR technique"
1
RAZA, ABIDA, and NAUREEN A KHATTAK. "REAL TIME PCR;." Professional Medical Journal 19, no. 06 (November 3, 2012): 751–59. http://dx.doi.org/10.29309/tpmj/2012.19.06.2455.
Full textAbstract:
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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Saienko, Artem, Mykyta Peka, Viktor Balatsky, Sergii Korinnyi, and Oleksandr Tsereniuk. "GMO analysis technique based on PCR and real-time PCR methods." Pig breeding the interdepartmental subject scientific digest, no. 75-76 (December 7, 2021): 58–67. http://dx.doi.org/10.37143/0371-4365-2021-75-76-06.
Full textAbstract:
The use of genetically modified organisms (GMOs) is promising for overcoming the shortage of food products in the world and solving the problem of hunger arising in various regions. At the same time, the use of GMOs has become a cause of debate, as the safety of consuming GMO products for human health remains unproven. The risks associated with GMOs cause public concern, which has led to the restriction of the use of GMOs and their products in many countries and the need for constant control of their content in food products. This study describes methods based on the polymerase chain reaction (PCR), such as Real-time PCR and PCR with electrophoretic separation of amplificates, which are generally accepted in GMO analysis. In order to control the presence of GMOs in food raw materials, feed and finished products of plant and animal origin, screening is carried out for the presence of the most common genetic engineering structures used during the creation of GMOs: CaMV 35S promoter and NOS terminator. Both Real-time PCR and PCR with electrophoretic separation of amplificates allow to establish the presence of GMOs with high accuracy, and Real-time PCR is also used to determine the concentration of GMOs in the studied samples. The work presents a typical electrophoresis with visualization of the obtained PCR fragments that required electrophoretic separation and fragments that were synthesized in the Real-time PCR reaction, and determined the approximate sizes of the obtained fragments relative to the pBR322 DNA-MspI molecular weight marker. The approach described in this study, based on the use of PCR techniques, can be successfully used for GMO analysis of all groups of raw materials and finished products of animal and plant origin, and is also well adapted for the detection of various genetic engineering structures, not limited to the CaMV 35S promoter and terminator NOS, which makes it possible to increase the efficiency of such analysis and continue its application in relation to new genetic engineering structures used during the creation of GMOs. Keywords: genetically modified organisms (GMOs), PCR methods, GMO analysis, raw materials and finished products of plant and animal origin
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Skitovich, G. S., N. B. Shadrova, O. V. Pruntova, and K. V. Serova. "REAL-TIME PCR OPTIMIZATION FOR LISTERIA MONOCYTOGENES GENOME DETECTION." Veterinary Science Today, no. 3 (October 3, 2018): 63–68. http://dx.doi.org/10.29326/2304-196x-2018-3-26-63-68.
Full textAbstract:
Listeria monocytogenes is one of the major food contaminants causing the illness, called Listeriosis. Listeriosis incidence is much less, than the number salmonellosis and campylobacteriosis cases, but the clinical disease is significantly more severe and has a higher mortality. That’s why the development of species-specific PCR techniques to detect L. monocytogenes genome is a topical task. L. monocytogenes bacteria genome detection technique using real-time polymerase chain reaction (qRT-PCR) was improved. The amplification target was a highly specific and suitable for qualification of all strains iap gen, coding L. monocytogenes р60 surface protein. Optimum magnesium concentration (6 mM) and primer annealing temperature (57 °С) were selected. The sensitivity and specificity of the technique were identified. Detection threshold was 120 target molecules. The results obtained demonstrate that optimized qRT-PCR version, based on iap gen amplification, enables to detect L. monocytogenes in animal product and food samples. Optimized qRT-PCR-based screening tests ensure rapid and reliable results.
4
Burggraf, Siegfried, and Bernhard Olgemöller. "Simple Technique for Internal Control of Real-Time Amplification Assays." Clinical Chemistry 50, no. 5 (May 1, 2004): 819–25. http://dx.doi.org/10.1373/clinchem.2003.027961.
Full textAbstract:
Abstract Background: In real-time PCR assays, the most accurate way to identify false-negative results, e.g., those caused by PCR inhibitors, is to add to samples an internal control that will be coamplified with the target (e.g., pathogen) DNA. Current internal control procedures, however, which usually involve the introduction of a DNA fragment, are complex, time-consuming, and expensive. Methods: Single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time PCR assays. Mismatches were included in the probe-binding region of the internal control oligonucleotide (ICO) to prevent probe–control hybridization during the fluorescence acquisition step of the PCR. Amplified ICOs were detected by melting point analysis. ICOs could be added directly to the sample material before DNA extraction. Results: To demonstrate the feasibility of the new approach, we designed ICOs for the LightCycler hybridization probe assays for Mycobacterium tuberculosis complex, hepatitis B virus, herpes simplex virus, and varicella zoster virus. In each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis. Conclusions: A single-stranded oligonucleotide that mimics the target region of the pathogen but is clearly distinguishable from the target during melting point analysis can serve as a simple, cost-effective internal control for real-time amplification assays. Such control oligonucleotides are easy to design and inexpensive. A costly second probe system is not necessary. Moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications.
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Bębnowska, Dominika, Rafał Hrynkiewicz, and Paulina Niedźwiedzka-Rystwej. "Real-Time PCR Confirms Infection with Lagovirus europaeus." Applied Sciences 11, no. 2 (January 11, 2021): 656. http://dx.doi.org/10.3390/app11020656.
Full textAbstract:
Lagovirus europaeus GI.1/GI.2 is an etiological agent causing the highly dangerous rabbit hemorrhagic disease (RHD). Molecular research is the basic tool today that can help solve epidemic problems related to the expansion of pathogens in the world. By using the real-time polymerase chain reaction technique (PCR), we detected three different strains of Lagovirus europaeus/GI.1, which is an RNA virus infecting mainly rabbits. The results showed that the method used was fast, very specific, and effective.
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Bębnowska, Dominika, Rafał Hrynkiewicz, and Paulina Niedźwiedzka-Rystwej. "Real-Time PCR Confirms Infection with Lagovirus europaeus." Applied Sciences 11, no. 2 (January 11, 2021): 656. http://dx.doi.org/10.3390/app11020656.
Full textAbstract:
Lagovirus europaeus GI.1/GI.2 is an etiological agent causing the highly dangerous rabbit hemorrhagic disease (RHD). Molecular research is the basic tool today that can help solve epidemic problems related to the expansion of pathogens in the world. By using the real-time polymerase chain reaction technique (PCR), we detected three different strains of Lagovirus europaeus/GI.1, which is an RNA virus infecting mainly rabbits. The results showed that the method used was fast, very specific, and effective.
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Christopher-Hennings, Jane, Matthew A. Dammen, Shelleen R. Weeks, William B. Epperson, Shri N. Singh, Gina L. Steinlicht, Ying Fang, et al. "Comparison of Two DNA Extractions and Nested PCR, Real-Time PCR, a New Commercial PCR Assay, and Bacterial Culture for Detection of Mycobacterium Avium Subsp. Paratuberculosis in Bovine Feces." Journal of Veterinary Diagnostic Investigation 15, no. 2 (March 2003): 87–93. http://dx.doi.org/10.1177/104063870301500201.
Full textAbstract:
In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.
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Artika, I. Made, Yora Permata Dewi, Ita Margaretha Nainggolan, Josephine Elizabeth Siregar, and Ungke Antonjaya. "Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis." Genes 13, no. 12 (December 16, 2022): 2387. http://dx.doi.org/10.3390/genes13122387.
Full textAbstract:
Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.
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Yang, Wenli, H. D. Alan Lindquist, Vitaliano Cama, Frank W. Schaefer, Eric Villegas, Ronald Fayer, Earl J. Lewis, Yaoyu Feng, and Lihua Xiao. "Detection of Toxoplasma gondii Oocysts in Water Sample Concentrates by Real-Time PCR." Applied and Environmental Microbiology 75, no. 11 (April 10, 2009): 3477–83. http://dx.doi.org/10.1128/aem.00285-09.
Full textAbstract:
ABSTRACT PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.
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Valero, Clara, Laura de la Cruz-Villar, Óscar Zaragoza, and María José Buitrago. "New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections." Journal of Clinical Microbiology 54, no. 12 (September 14, 2016): 2910–18. http://dx.doi.org/10.1128/jcm.01580-16.
Full textAbstract:
The diagnosis of invasive fungal infections (IFIs) is usually based on the isolation of the fungus in culture and histopathological techniques. However, these methods have many limitations often delaying the definitive diagnosis. In recent years, molecular diagnostics methods have emerged as a suitable alternative for IFI diagnosis. When there is not a clear suspicion of the fungus involved in the IFI, panfungal real-time PCR assays have been used, allowing amplification of any fungal DNA. However, this approach requires subsequent amplicon sequencing to identify the fungal species involved, increasing response time. In this work, a new panfungal real-time PCR assay using the combination of an intercalating dye and sequence-specific probes was developed. After DNA amplification, a melting curve analysis was also performed. The technique was standardized by using 11 different fungal species and validated in 60 clinical samples from patients with proven and probable IFI. A melting curve database was constructed by collecting those melting curves obtained from fungal species included in the standardization assay. Results showed high reproducibility (coefficient of variation [CV] < 5%; r > 0.95) and specificity (100%). The overall sensitivity of the technique was 83.3%, with the group of fungi involved in the infection detected in 77.8% of the positive samples with IFIs covered by molecular beacon probes. Moreover, sequencing was avoided in 67.8% of these “probe-positive” results, enabling report of a positive result in 24 h. This technique is fast, sensitive, and specific and promises to be useful for improving early diagnosis of IFIs.
Dissertations / Theses on the topic "Real-Time PCR technique"
1
Lintell, Nicholas Adrian, and n/a. "DNA Aberrations in Atypical Cancer Cohorts." Griffith University. School of Biomolecular and Biomedical Science, 2006. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20061009.164402.
Full textAbstract:
The incidence of Squamous Cell Carcinoma is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 45% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis lesion and with a small but significant chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. This study had an explicit emphasis on the mitochondrial genome and nuclear genes that encode for subunits in the mitochondrial regulated energy transducing oxidative phosphorylation pathways. More specifically the first aim of this project was to analyse the NDUFA8, PTCH, NDUFAS, SMOH, SDHD, MMPI2, NDUFV1, EMSI, COXVIIc, and RASAI genes via non-specific fluorophoric Real-Time PCR for genetic aberrations in an affected Solar Keratosis and control cohort. The second aim was to analyse two specific genes, SDHD and MMPI2, for copy number aberrations via Dual-Labelled Probe Real-Time PCR in the same affected Solar Keratosis and control cohort. The third aim was to analyse Mitochondrial DNA Depletion syndrome (MDS) in a chemically exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR. The significance of these studies is in their contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. Furthermore, it would determine whether the genes analysed in this study exist in greater prevalence in the affected Solar Keratosis population compared to the control cohort. With regard to the MDS component, identifying the presence of this disease in these individuals was initially undertaken as part of a study to provide evidence in compensation claims. The diagnosis may assist in their medical therapy, insofar as some of them were now suffering from liver malfunctions and atypical male breast cancer. Another application of this effective and low cost method of diagnosing MDS is in populations with high HTV incidences. This is due to the fact that the most common drug used to treat this disease can give rise to the expression of MDS, thus further complicating the health status of HIV infected individuals. The analysis of this research was accomplished via the Real-Time PCR technique, with a non-specific fluorophore component in addition to specific Dual-Labelled Probe components, to ascertain the general nature of any aberration identified in the sample cohort. This project also employed additional methods of analysis such as DHPLC and DNA sequencing to assist in determining the veracity of its aims, particularly in terms of the precise detection of genetic aberrations via Real-Time PCR. Patients exhibiting male breast cancer and liver malftinctions were also analysed via Dual-Labelled Probe RealTime PCR to ascertain the presence of Mitochondrial DNA Depletion syndrome, a disorder characterised by lactic acidosis, liver failure, seizures, and congestive heart failure. Determining the presence of this syndrome in these patients would assist in their medical treatment, and contribute to the analytical methods available to diagnose this syndrome, which is known to occur in HIV sufferers due to the nucleoside drugs used to combat the disease. Real-Time PCR can adequately gauge the integrity of a genetic area in terms of amplicon malformities (non-specific-fluorophoric) and DNA copy number aberrations (Dual-Labelled Probe) via fluorophore signal differentials compared to wild-type samples and housekeeper profiles. The results of the first component of this project, namely the analysis of five gene pairs by non-specific fluorophoric Real-Time PCR, highlighted that a significantly higher incidence of putative aberrants is evident in the affected population when compared to the control cohort. The genes analysed were NDUFA8, PTCH, NDUFA5, SMOH, SDHD, MMP 12, NDUFVI, EMS 1, COXVIIc, and RASA 1. These ten genes were subdivided into five pairs; one of the pair being a gene associated with the development of a non-melanotic skin cancer (NMSC), the other a gene encoding for a subunit of the Electron Transport Chain (ETC). Each of these pairs exists in close proximity to one another on a particular chromosomal locale. Differences were highlighted in the single gene triplicate run population. The ETC genes (NDUFA8, NDUFA5, SDHD, NIDUFVI, COXVIIc) exhibited 10 / 720 (1.37%) as being putative mutants in the control population, compared to 117 / 675 (17.3%) for the affected population (p value less than 0.0001). The NMSC gene analysis (PTCH, SMOH, MMPI2, EMSI, RASA1) produced a 16 / 720 (2.22%) ratio for the control population, with the affected population having an incidence of 97 / 675 (14.4 %) for putative mutants (p value less than 0.0001). The observance of putative aberrants in the NDUFVI (p less than 0.018), EMS1 (p less than 0.003), COXVTIc (p less than 0.001), and RASA I (p less than 0.009) genes in the affected Solar Keratosis (SK) population was significantly higher than that observed in the control population. The majority of aberrations detected via the non-specific fluorophoric Real-Time PCR technique were small nucleotide base insertions and deletions. The analysis of the SK affected and control cohort via Real-Time PCR proved a cost-effective and reliable method in identifying the presence of DNA aberrations such as non-instructional sites. The results of the second component extended the findings of the non-specific fluorophoric analysis. The SDHD and MMPI 2 genes were analysed for copy number aberrations via Dual-Labelled Probe Real-Time PCR for genetic aberrations the same affected and control Solar Keratosis cohort. It was found that 12 of 279 samples had identifiable copy-number aberrations in either the SDHD or MMPI2 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The MMPI2 gene also had nine samples identified as possessing an intronic heterozygous base-pair substitution anomaly via DNA sequencing. The NDUFA8 gene had 12 samples identified as anomalous via the DHPLC technique, 11 of which were identified via non-specific fluorophoric Real-Time PCR, with the analysis performed to verify the accuracy of the Real-Time technique in identifying DNA aberrations. This study identified DNA aberrations in an affected Solar Keratosis and control cohort and ascertained several particular genomic abnomialities in the SDHD, MMPI2 and NDUFA8 genes, with an emphasis on copy-number aberrations and amplicon abnormalities. In the third component of this study, namely the analysis of Mitochondrial DNA Depletion syndrome (MDS) in a jet-fuel exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR, the results indicated that four of the seven patients were expressing MDS. Of the four patients who exhibited a reduction in mitochondrial copy-number the average decrease was of a four-fold level, or approximately a depletion of mitochondrial copies from 200 plus to ~ 54 (74 % reduction in MtDNA). The patients who contributed DNA for investigation into the presence of MDS were suffering from liver malfunction and atypical male breast cancer. The Dual-Labelled Probe technique proved a reliable and cost effective method in identifying the presence of MDS in these patients, with the DNA extracted from fresh white blood cells that had been isolated using the Ficoll-Hypaque method. The importance of this is that accurate levels of Mitochondrial DNA copy numbers can be ascertained in white blood cells as it removes the presence of platelets, which also contain mitochondria but no nucleus. The analysis of ETC and NMSC associated genes in addition to mitochondrial copy number integrity means that this study investigated two aspects of the carcinogenetic pathway i.e. abnormal energy regulation and the regulation of micromolecular and macromolecular cellular homeostatic mechanisms. The mechanism of programmed cell death or apoptosis is regulated by the mitochondria and the ability of a genetically damaged cell to evade the apoptotic process is directly linked to a cell becoming cancerous. It is only after the evasion of apoptosis and the replication of the damaged cells' DNA into daughter cells that neoplastic events can occur. Thus, this study contributed to the understanding of how neo-plastic lesions may develop and progress into invasive tumours. It additionally assisted in proving the effectiveness of the RealTime PCR technique in detecting DNA aberrations and mitochondrial copy number anomalies.
2
Lintell, Nicholas Adrian. "DNA Aberrations in Atypical Cancer Cohorts." Thesis, Griffith University, 2006. http://hdl.handle.net/10072/365589.
Full textAbstract:
The incidence of Squamous Cell Carcinoma is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 45% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis lesion and with a small but significant chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. This study had an explicit emphasis on the mitochondrial genome and nuclear genes that encode for subunits in the mitochondrial regulated energy transducing oxidative phosphorylation pathways. More specifically the first aim of this project was to analyse the NDUFA8, PTCH, NDUFAS, SMOH, SDHD, MMPI2, NDUFV1, EMSI, COXVIIc, and RASAI genes via non-specific fluorophoric Real-Time PCR for genetic aberrations in an affected Solar Keratosis and control cohort. The second aim was to analyse two specific genes, SDHD and MMPI2, for copy number aberrations via Dual-Labelled Probe Real-Time PCR in the same affected Solar Keratosis and control cohort. The third aim was to analyse Mitochondrial DNA Depletion syndrome (MDS) in a chemically exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR. The significance of these studies is in their contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. Furthermore, it would determine whether the genes analysed in this study exist in greater prevalence in the affected Solar Keratosis population compared to the control cohort. With regard to the MDS component, identifying the presence of this disease in these individuals was initially undertaken as part of a study to provide evidence in compensation claims. The diagnosis may assist in their medical therapy, insofar as some of them were now suffering from liver malfunctions and atypical male breast cancer. Another application of this effective and low cost method of diagnosing MDS is in populations with high HTV incidences. This is due to the fact that the most common drug used to treat this disease can give rise to the expression of MDS, thus further complicating the health status of HIV infected individuals. The analysis of this research was accomplished via the Real-Time PCR technique, with a non-specific fluorophore component in addition to specific Dual-Labelled Probe components, to ascertain the general nature of any aberration identified in the sample cohort. This project also employed additional methods of analysis such as DHPLC and DNA sequencing to assist in determining the veracity of its aims, particularly in terms of the precise detection of genetic aberrations via Real-Time PCR. Patients exhibiting male breast cancer and liver malftinctions were also analysed via Dual-Labelled Probe RealTime PCR to ascertain the presence of Mitochondrial DNA Depletion syndrome, a disorder characterised by lactic acidosis, liver failure, seizures, and congestive heart failure. Determining the presence of this syndrome in these patients would assist in their medical treatment, and contribute to the analytical methods available to diagnose this syndrome, which is known to occur in HIV sufferers due to the nucleoside drugs used to combat the disease. Real-Time PCR can adequately gauge the integrity of a genetic area in terms of amplicon malformities (non-specific-fluorophoric) and DNA copy number aberrations (Dual-Labelled Probe) via fluorophore signal differentials compared to wild-type samples and housekeeper profiles. The results of the first component of this project, namely the analysis of five gene pairs by non-specific fluorophoric Real-Time PCR, highlighted that a significantly higher incidence of putative aberrants is evident in the affected population when compared to the control cohort. The genes analysed were NDUFA8, PTCH, NDUFA5, SMOH, SDHD, MMP 12, NDUFVI, EMS 1, COXVIIc, and RASA 1. These ten genes were subdivided into five pairs; one of the pair being a gene associated with the development of a non-melanotic skin cancer (NMSC), the other a gene encoding for a subunit of the Electron Transport Chain (ETC). Each of these pairs exists in close proximity to one another on a particular chromosomal locale. Differences were highlighted in the single gene triplicate run population. The ETC genes (NDUFA8, NDUFA5, SDHD, NIDUFVI, COXVIIc) exhibited 10 / 720 (1.37%) as being putative mutants in the control population, compared to 117 / 675 (17.3%) for the affected population (p value less than 0.0001). The NMSC gene analysis (PTCH, SMOH, MMPI2, EMSI, RASA1) produced a 16 / 720 (2.22%) ratio for the control population, with the affected population having an incidence of 97 / 675 (14.4 %) for putative mutants (p value less than 0.0001). The observance of putative aberrants in the NDUFVI (p less than 0.018), EMS1 (p less than 0.003), COXVTIc (p less than 0.001), and RASA I (p less than 0.009) genes in the affected Solar Keratosis (SK) population was significantly higher than that observed in the control population. The majority of aberrations detected via the non-specific fluorophoric Real-Time PCR technique were small nucleotide base insertions and deletions. The analysis of the SK affected and control cohort via Real-Time PCR proved a cost-effective and reliable method in identifying the presence of DNA aberrations such as non-instructional sites. The results of the second component extended the findings of the non-specific fluorophoric analysis. The SDHD and MMPI 2 genes were analysed for copy number aberrations via Dual-Labelled Probe Real-Time PCR for genetic aberrations the same affected and control Solar Keratosis cohort. It was found that 12 of 279 samples had identifiable copy-number aberrations in either the SDHD or MMPI2 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The MMPI2 gene also had nine samples identified as possessing an intronic heterozygous base-pair substitution anomaly via DNA sequencing. The NDUFA8 gene had 12 samples identified as anomalous via the DHPLC technique, 11 of which were identified via non-specific fluorophoric Real-Time PCR, with the analysis performed to verify the accuracy of the Real-Time technique in identifying DNA aberrations. This study identified DNA aberrations in an affected Solar Keratosis and control cohort and ascertained several particular genomic abnomialities in the SDHD, MMPI2 and NDUFA8 genes, with an emphasis on copy-number aberrations and amplicon abnormalities. In the third component of this study, namely the analysis of Mitochondrial DNA Depletion syndrome (MDS) in a jet-fuel exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR, the results indicated that four of the seven patients were expressing MDS. Of the four patients who exhibited a reduction in mitochondrial copy-number the average decrease was of a four-fold level, or approximately a depletion of mitochondrial copies from 200 plus to ~ 54 (74 % reduction in MtDNA). The patients who contributed DNA for investigation into the presence of MDS were suffering from liver malfunction and atypical male breast cancer. The Dual-Labelled Probe technique proved a reliable and cost effective method in identifying the presence of MDS in these patients, with the DNA extracted from fresh white blood cells that had been isolated using the Ficoll-Hypaque method. The importance of this is that accurate levels of Mitochondrial DNA copy numbers can be ascertained in white blood cells as it removes the presence of platelets, which also contain mitochondria but no nucleus. The analysis of ETC and NMSC associated genes in addition to mitochondrial copy number integrity means that this study investigated two aspects of the carcinogenetic pathway i.e. abnormal energy regulation and the regulation of micromolecular and macromolecular cellular homeostatic mechanisms. The mechanism of programmed cell death or apoptosis is regulated by the mitochondria and the ability of a genetically damaged cell to evade the apoptotic process is directly linked to a cell becoming cancerous. It is only after the evasion of apoptosis and the replication of the damaged cells' DNA into daughter cells that neoplastic events can occur. Thus, this study contributed to the understanding of how neo-plastic lesions may develop and progress into invasive tumours. It additionally assisted in proving the effectiveness of the RealTime PCR technique in detecting DNA aberrations and mitochondrial copy number anomalies.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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Netshikweta, Rembuluwani. "Optimisation and assessment of real-time PCR techniques for the detection of selected food- and waterborne viruses." Diss., University of Pretoria, 2011. http://hdl.handle.net/2263/24883.
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The transmission of human pathogens by faecally contaminated fruit and vegetables is well established, but the burden of disease caused by foodborne pathogens is unknown. Fresh produce can be contaminated through the use of polluted irrigation water or by the handling of the produce by infected individuals either pre- or post harvest. There is very little known regarding the extent of viral contamination of irrigation water and fresh produce in South Africa. Noroviruses (NoV) and hepatitis A virus (HAV) are recognized as leading causes of foodborne viral disease. These viruses are transmitted predominantly via the faecal–oral route, primarily person-to-person by direct contact with an infected person, or indirectly by ingestion of contaminated food and water. The detection of enteric viruses in food or water is problematical and complex as many foodborne viruses, including HAV and NoV, cannot be readily isolated in cell culture. The aim of this investigation was to develop and optimise simple and efficient methods for the concentration and detection of NoV GII and HAV in irrigation water and fresh produce. These methods would then be applied to field samples of irrigation water and fresh produce to try and establish a link between viral contamination detected in irrigation water and that on associated irrigated fresh produce. The efficiency of different commercial real-time reverse transcriptase-polymerase chain reaction amplification kits for the realtime detection of HAV, NoV GI and NoV GII was assessed, and standard curves for the quantitative detection of these viruses were constructed using the most appropriate kit. Using two types of fresh produce, three different elution buffers, each at two pHs, with two different elution times were compared to establish which buffer was the most efficient for the extraction of viruses from the fresh produce. The tris-glycine beef extract buffer (pH 9.5) with an elution time of 20 minutes most efficient for the extraction of the selected enteric viruses from fresh produce. From April 2008 to November 2009, 86 irrigation water and 72 fresh produce samples were collected from commercial and subsistence farms, street vendors and commercial outlets. All the irrigation water and fresh produce samples were analysed for HAV, NoV GI and NoV GII. Overall, 16.3 % (13/86) and 12.5 % (9/72) of irrigation water and fresh produce samples tested positive for one or more human pathogenic viruses, namely NoV GII and HAV, respectively. Nucleotide sequence and phylogenetic analysis of the HAV and NoV GII strains identified clinically relevant viruses in the irrigation water and on the fresh produce. A direct link between contaminated irrigation water and contamination of fresh produce could not be established, but irrigation water was identified as a possible source of contamination of the fresh produce. The results also suggested that food handlers contributed significantly to the viral contamination of the fresh produce. This study highlights the potential health risk posed by fresh produce to consumers in South Africa and highlights the need for further in depth studies to quantify the risk to consumers. This study represents new data on the occurrence of enteric viruses in food and water in South Africa and is crucial for the development of effective intervention and control strategies for food safety in South Africa. CopyrightDissertation (MSc)--University of Pretoria, 2011.
Medical Virology
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Li, Sichu. "Application of Machine Learning Techniques for Real-time Classification of Sensor Array Data." ScholarWorks@UNO, 2009. http://scholarworks.uno.edu/td/913.
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There is a significant need to identify approaches for classifying chemical sensor array data with high success rates that would enhance sensor detection capabilities. The present study attempts to fill this need by investigating six machine learning methods to classify a dataset collected using a chemical sensor array: K-Nearest Neighbor (KNN), Support Vector Machine (SVM), Classification and Regression Trees (CART), Random Forest (RF), Naïve Bayes Classifier (NB), and Principal Component Regression (PCR). A total of 10 predictors that are associated with the response from 10 sensor channels are used to train and test the classifiers. A training dataset of 4 classes containing 136 samples is used to build the classifiers, and a dataset of 4 classes with 56 samples is used for testing. The results generated with the six different methods are compared and discussed. The RF, CART, and KNN are found to have success rates greater than 90%, and to outperform the other methods.
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Sylvester, John T. "Development and evaluation of new techniques to quantify ruminal pool size and duodenal flow of protozoal nitrogen." Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1126015772.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xviii, 131 p.; also includes graphics. Includes bibliographical references (p. 120-131). Available online via OhioLINK's ETD Center
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Smith, Shelle Ann. "A comparison of diagnostic techniques for detecting salmonella spp in equine fecal samples using culture methods, gel-based pcr, and real-time pcr assays." Thesis, Texas A&M University, 2003. http://hdl.handle.net/1969.1/5735.
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Salmonellae are enteric bacteria infecting animals and humans. Large animal clinics and Veterinary Teaching Hospitals are greatly affected by Salmonella outbreaks and nosocomial infection. The risk of environmental contamination and spread of infection is increased when animals are confined in close contact with each other and subjected to increased stress factors. This study was designed to compare double-enrichment culture techniques with Gel-based and Real-time PCR assays in the quest for improved diagnostic methods for detecting Salmonella in equine fecal samples. 120 fecal samples submitted to the Clinical Microbiology Laboratory of the Veterinary Medical Teaching Hospital at Texas A&M University (CML, VMTH, TAMU) were tested for Salmonella using all three techniques. Double-enrichment bacterial culture detected 29 positive results (24%), Real-time PCR detected 33 positive results (27.5%), and Gel-based PCR detected 73 positives results (60.8%). While culture and real-time PCR methods had similar results, the gel-based PCR method detected many more positive results, indicating probable amplicon contamination. Real-time PCR can be completed as soon as the day after submission while culture techniques may take 2 to 5 days to complete. However, viable bacterial cells are needed for antimicrobial susceptibility testing and serotyping: both important for epidemiological studies. Therefore, double-enrichment bacterial culture performed concurrently with real-time PCR methods could be efficient in clinical settings where both accurate and expedient results are required.
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Solliec, Laurent. "Real time flow rate modelling in disturbed conditions from velocity profilers." Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAD052.
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L'installation de systèmes de mesure est d'une utilisation cruciale pour la gestion des réseaux d'assainissement ou des canaux d 'irrigation. La plupart des structures gouvernementales ou privées ainsi que les agglomérations s'équipent de systèmes de mesure de débit afin de se conformer avec la législation européenne. La plupart des débitmètres fournissent des données en temps réel i.e. l'information est transmise en permanence. aux centrales d'acquisition pour une gestion de l'architecture du système de canaux. La mesure en canaux ouverts est souvent ultrasonore. L'objectif de cette thèse est de proposer une méthode en temps réel afin de corréler les vitesses locales en une vitesse moyenne dans les conditions observables par les utilisateurs en canaux ouverts. Les thématiques impliquées à cette étude sont multiples: les techniques de mesure, l'hydrodynamique en canaux ouverts représentée par la turbulence (ici plus particulièrement les courants secondaires), les lois de paroi, le nombre de Froude ... l'ensemble de ces thématiques doit être investi en canaux pleinement développés où les conditions sont stables dans l'espace mais aussi pour des conditions perturbées telles que les structures hétérogènes ou transitoires.La technique de mesure est un point clé: quelle est la technique la plus applicable aux conditions de mesure i.e. les canaux étroits? Les canaux étroits varient très rapidement en tem1es de taux de remplissage : la technique la plus adaptée est le profileur ultrasonique.La compréhension des effets hydrodynamiques est essentielle afin de développer un modèle de conversion. Les canaux droits sont influencés par l'hydrodynamique des écoulements, la géométrie mais aussi et principalement par leurs interactions. En canaux droits, les courants secondaires sont primordiaux même s'ils se traduisent par un effet le plus observable : le dip-phénomène, i.e. la présence d'un maximum de vitesse non pas à la surface d'eau mais en dessous pour les canaux étroits. Ces courants secondaires sont fortement sensibles au rapport d'aspect, la géométrie et la variabilité de la rugosité le long de la paroi, passablement sensible à la rugosité et indépendant du nombre de Froude .Les perturbations, à l'aval desquelles sont installés les débitmètres ultrasonores, sont majoritairement représentées par les coudes et les jonctions. Dans les coudes, les tourbillons sont liés aux forces centrifuges (gros tourbillon) et la turbulence (petit tourbillon). Pour les jonctions, les tourbillons diffèrent des deux précédentes configurations avec la présence à l'aval de la jonction de 3 tourbillons (due à un étirement des tourbillons par l'arrivée latérale). Les capteurs ne sont pas installés directement au niveau de la perturbation mais à l'aval. Dans la littérature, les distances requises pour retrouver des conditions proches de l'écoulement pleinement développé devraient excéder environ 50 hauteurs d'eau. En pratique, ces distances sont plus proches de5-10 fois la largeur du canal ou du tirant d'eau. L'application de modèle basée sur l'écoulement pleinement développé corrélé à un capteur n'est pas recommandableThe installation of flow rate measurement systems is an important factor in regard to the management of sewer and irrigation networks. Most cities and infrastructure succeed in obtaining sufficient flow measurements to satisfy European Regulation rules. Most flow meters comprise real time systems; this means that the information is permanently transferred to a data base for the management and optimization of the particular network. The measurement technology deployed is typically ultrasound based. Within the number of measurement points a high percentage are often deficient and create specific difficulties (>75% of Venturi flumes are inaccurate according to Anglian Water, a UK water and wastewater company). The study presented here focuses on flow meters which calculate discharge using measurement of level, cross sectional area and the correlation of local velocity to generate a mean value. The aim of this thesis is to propose a real time method to enable determination of this “conversion” under realistic configurations which Users find in open channels. The synthesis of measurement points through an understanding of hydraulic conditions (Bonakdari, 2006) provides a method to create flow data allowing local point velocities to be converted into an overall mean value. The approach has limitations and may fail in industrial situations but can be used for very complex configurations. It also requires specialists with knowledge of the technique who are rarely available to Users. What is proposed here is an alternative method to Bonakdari for simpler configurations. The aim is to evaluate the flow rate with acceptable accuracy using these technics and to establish a relationship between local velocities and the mean velocity according to Regulatory requirements (8% are required in UK, 5 to 8% in Germany depending on area). The individual components are here: the measurement techniques; the hydrodynamics represented with the turbulence (secondary currents in open channels); the wall / roughness effects; the Froude number … for fully developed conditions where conditions become stable in space but for disturbed conditions, as well such as heterogeneous structures or transition conditions
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Munõz, Vanessa Nathalia Vargas. "Detection and quantification of Colletotrichum abscissum from leaves of budwood increase block and citrus nursery plants by real time PCR." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/11/11135/tde-22112018-154045/.
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Brazil is the largest citrus producer in the world and has a large global citrus market share. However, several diseases affect the crop, being postbloom fruit drop (PFD) one of them. PFD has gained importance in São Paulo State for the displacement of citrus areas to regions with weather conditions more favorable for this disease. The accurate identification of the causal agent of the PFD has been performed and it was renamed as Colletotrichum abscissum. The origin of the initial inoculum is still an enigma for PFD epidemics and the hypotheses that the initial inoculum could be present in propagation material have been discussed but it has never been demonstrated. The objective of this work was to detect and quantify Colletotrichum abscissum from citrus leaves of budwood increase block and citrus nursery plants by qPCR. Four commercial citrus farms from São Paulo State, Brazil with budwood increase block and citrus nursery plants of Pera and Valencia sweet orange varieties were used for this work. C. abscissum was detected in budwood increase block and in nursery plant in both varieties (Valencia and Pera) at the four farms sampled. Out of 122 budwood increase block samples, 89 (73%) were positive for C. absicissum. From nursery plants, out of 175 samples, 129 (73%) were detected with the pathogen. The majority of the positive samples of budwood increase blocks and nursery plants contained 10 to 200 and 10 to 400 conidia of C. absicissum, respectively. With the methods used was not possible to isolate the fungus from vegetative material. This finding suggests a new long distances dispersion type of C. abscissum in the cycle of postbloom fruit drop by propagation material. Confirmation of C. abscissum in budwood increase block and nursery plants would lead to update regulations for the production of certified citrus nursery trees and searching for new control strategies of the pathogen.O Brasil é o maior produtor de citros do mundo e possui uma grande participação no mercado global de citros. No entanto, várias doenças afetam a cultura, sendo uma delas a podridão floral dos citros (PFC). PFC ganhou importância no Estado de São Paulo pelo deslocamento de áreas de citros para regiões com condições climáticas mais favoráveis para a doença. A identificação precisa do agente causal do PFC foi realizada, tendo sido renomeado como Colletotrichum abscissum. A origem do inóculo inicial ainda é um enigma para as epidemias de PFC e as hipóteses do que o inóculo inicial poderia estar presente no material de propagação já foram discutidas, mas nunca foram demonstradas. O objetivo deste trabalho foi detectar e quantificar Colletotrichum abscissum em folhas de borbulheiras e mudas de citros por meio de qPCR. Neste trabalho, foram utilizadas quatro fazendas comerciais de citros do Estado de São Paulo, Brasil, com borbulheiras e viveiros de mudas de citros das variedades laranja Pera e Valência. C. abscissum foi detectado em borbulheiras e em mudas em ambas as variedades (Valência e Pêra) nas quatro fazendas amostradas. Das 122 amostras de folhas de borbulheiras, 89 (73%) foram positivas para C. absicissum. Das 175 amostras de folhas de mudas de citros, 129 (73%) foram detectadas com o patógeno. A maioria das amostras positivas de borbulheiras e mudas de citros continham 10 a 200 e 10 a 400 conídios de C. absicissum, respectivamente. Com os métodos utilizados, não foi possível isolar o fungo do material vegetativo. Esta descoberta sugere um novo tipo de dispersão a longas distâncias de C. abscissum no ciclo de podridão floral dos citros por meio do material de propagação. A confirmação de C. abscissum nas borbulheiras e mudas de citros levaria à atualização da regulamentação para a produção de mudas de citros certificadas e à busca de novas estratégias de controle do patógeno.
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Andrade, Thales Passos de. "DEVELOPMENT AND APPLICATION OF NOVEL QUANTITATIVE AND QUALITATIVE MOLECULAR TECHNIQUES FOR DETECTION OF INFECTIOUS MYONECROSIS VIRUS (IMNV) IN PACIFIC WHITE SHRIMP LITOPENAEUS VANNAMEI." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/195726.
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Infectious myonecrosis, caused by infectious myonecrosis virus (IMNV), is an important disease of shrimp that has adversely affected the production of cultured Litopenaeus vannamei. The studies reported here were centered on development and/or validation of alternative diagnostic methods for detection of IMNV. Hence, two manuscripts were published in the Journal of Aquaculture and one manuscript was published in the Journal of Fish Diseases. Chapter 2 describes the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe. The results showed that this real-time RT-PCR assay can detect as little as 10 IMNV copies/μl RNA, while the nested RT-PCR can detect 1000 copies/μl RNA. These findings suggest that the TaqMan real-time RT-PCR is “the gold standard” for screening shrimp to protect aquaculture production systems from losses caused by IMNV, because it provides quantification, higher sensitivity and specificity, and because it is less time consuming and less prone to contamination compared to conventional gel-based RT-PCR. In Chapter 3 I evaluated if prolonged storage of infectious myonecrosis virus (IMNV) infected shrimp in Davidson’s AFA fixative can degrade its double-stranded RNA genome resulting in false negative ISH reactions. Shrimp were collected at Day 12 post-injection and fixed in Davidson’s AFA for five different preservation times (1, 2, 4, 7 and 10 days). Hence, in the present report it was found that the length of time (up to 10 days) in Davidson’s AFA did not have a deleterious effect on the ISH reaction for IMNV. The Chapter 4 describes the development of a reverse transcription loop-mediated isothermal amplification and nucleic acid lateral flow (RT-LAMP-NALF) for detection of IMNV. The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The RT-LAMP-NALF was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.
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Catalá, García Santiago. "Development of DNA massive sequencing techniques and Real-Time PCR for the detection, identification and quantitation of Phytophthora spp. in environmental samples." Doctoral thesis, Universitat Politècnica de València, 2017. http://hdl.handle.net/10251/90644.
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In recent years the increase of global plant trade and human movement has promoted the risk of introduction of invasive plants and exotic pathogens. Biological invasions operate globally and are considered to be the second cause of biodiversity loss after direct habitat alteration and destruction. In this context, Phytophthora is one of the most important and aggressive plant pathogen in agriculture and forestry. Early detection and identification of its pathways are of high importance to minimize the threat that they pose to natural ecosystems. Different molecular-based methods, including real-time PCR and Next Generation Sequencing, have been developed and applied for the detection of plant pathogens in environmental samples. These methods allow fast and accurate pathogen detection and identification even when the inoculum amount is low. Therefore, the main objective of this thesis was the development of a new method for Phytophthora detection in environmental samples starting from extraction of environmental DNA (eDNA) from different sources (soil, roots and water) and different ecosystems. Different studies have applied High Throughput Sequencing (HTS) for the detection of Phytophthora species in soil samples, but not, to date, for water.
In the Chapter 3, genus-specific primers were adapted to assess Phytophthora species diversity in natural ecosystems using high-throughput amplicon pyrosequencing of eDNA from soil and water environments, based in the polymorphic and widely accepted barcoding target Internal Transcribed Spacer 1 (ITS1). The assay was validated with a control reaction with DNA of pure cultures. The objectives raised and developed of this study were: a) as main objective, development and application of HTS (High Throughput Sequencing) of Phytophthora-specific PCR amplicons to investigate the presence of Phytophthora in soil samples from different plant communities in natural forests, plantations and aquatic environments in the north of Spain; b) optimization of the conditions for emPCR amplification in order to obtain the best results in the pyrosequencing run; c) development of a bioinformatics pipeline for NGS data, focusing in the optimization of a barcoding threshold value to separate Molecular Operational Taxonomic Units (MOTUs).
Different score coverage threshold values were tested for optimal Phytophthora species separation in the bioinformatics analyses. Clustering at 99 % was the best criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams did not show significant matches to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems.
Once the technique was developed and validated, another objective was proposed in Chapter 2, focused on the oak decline. The evergreen holm oak (Quercus ilex) is the most representative tree species in the Iberian Peninsula and the main tree in oak-rangeland ecosystems (dehesas). Oak decline in non-calcareous soils in south-western Spain has been associated with Phytophthora cinnamomi for decades. However, other Phytophthora species such as P. quercina and P. psychrophila have been associated with Quercus decline in the eastern part of Spain where calcareous soils are predominant. With the aim of investigating the involvement of Phytophthora spp. in oak decline in eastern Spain, two forests in different geographical areas (Alcoi and Vallivana) were selected as sampling sites. Soil and root samples were analysed in parallel by amplicon pyrosequencing and real-time PCR. Metabarcoding analyses showed PhytophthoraEn los últimos años, el aumento del comercio mundial de plantas y el movimiento humano ha promovido el riesgo de introducción de plantas invasoras y patógenos exóticos. Las invasiones biológicas operan a nivel mundial y se consideran como la segunda causa de pérdida de biodiversidad después de la alteración y destrucción directas del hábitat. En este contexto, Phytophthora es uno de los patógenos vegetales más importantes y agresivos en la agricultura y la silvicultura. La detección temprana y la identificación de sus vías son de gran importancia para minimizar la amenaza que representan para los ecosistemas naturales. Se han desarrollado y aplicado diferentes métodos moleculares para la detección de patógenos de plantas en muestras ambientales. Estos métodos permiten una detección e identificación de patógenos rápida y precisa incluso cuando la cantidad de inóculo es baja. Por lo tanto, se propone un nuevo método mejorado para su detección en muestras ambientales a partir de la extracción de ADN ambiental (eDNA) de diferentes fuentes (suelo, raíces y agua) y diferentes ecosistemas. El objetivo del primer capítulo fue aplicar HTS (High Throughput Sequencing) para investigar la presencia de Phytophthora en diferentes comunidades de plantas en bosques naturales, plantaciones y ambientes acuáticos en el norte de España. El eDNA se extrajo del suelo y del agua de los ríos y arroyos de los bosques de Fagus sylvatica y Abies alba y de plantaciones de Chamaecyparis lawsoniana y Pseudotsuga menziesii en el norte de España (bosque de Irati y Villanúa). Se diseñó y aplicó un ensayo específico para la detección de Phytophthora mediante la secuenciación masiva de amplicones basado en la región ITS1. Diferentes valores de threshold se analizaron para la separación óptima de especies de Phytophthora en los análisis bioinformáticos. El agrupamiento al 99% fue el mejor criterio para separar la mayor parte de las especies de Phytophthora. Múltiples Unidades Operacionales Taxonómicas Moleculares (MOTU) correspondientes a 36 especies distintas de Phytophthora se amplificaron en las muestras ambientales. La pirosequenciación de amplicones de muestras de suelo reveló una diversidad baja de Phytophthora (13 especies) en comparación con las 35 especies detectadas en muestras de agua. Trece de los MOTU detectados en los ríos y arroyos no mostraron homología con secuencias depositadas en las bases de datos, lo que revela que la pirosequenciación del ADN ambiental es una estrategia útil para evaluar la diversidad de especies Phytophthora en los ecosistemas naturales. Una vez que la técnica fue desarrollada y validada, se propuso otro objetivo enfocado en el decaimiento de la carrasca. La carrasca (Quercus ilex) es la especie arbórea más representativa de la Península Ibérica y el árbol principal de las dehesas. El decaimiento de la carrasca en suelos no calcáreos en el suroeste de España se ha asociado con Phytophthora cinnamomi durante décadas. Sin embargo, otras especies de Phytophthora como P. quercina y P. psychrophila se han asociado con el declive de Quercus en la parte oriental de España donde predominan los suelos calcáreos. Con el objetivo de investigar la implicación de Phytophthora spp. en el declive de la carrasca en el este de España, se seleccionaron dos bosques en diferentes zonas geográficas (Alcoi y Vallivana) como lugares de muestreo. Las muestras de suelo y raíz se analizaron por pirosequenciación de amplicones. Los resultados de la secuenciación masiva mostraron la diversidad de especies de Phytophthora, y reveló que un taxón nunca aislado de Phytophthora, llamado provisional Phytophthora taxon ballota, fue la especie predominante en ambas áreas. Además, se desarrolló un ensayo de PCR a tiempo real, basado en los resultados de la pirosequenciación, para la detección de este taxón de Phytophthora nunca aislado, y también para la detección de P. quercina
En els últims anys, l'augment del comerç mundial de plantes i el moviment humà ha promogut el risc d'introducció de plantes invasores i patògens exòtics. Les invasions biològiques operen a nivell mundial i es consideren de com la segona causa de pèrdua de biodiversitat després de l'alteració i destrucció directa de l'hàbitat. En aquest context, Phytophthora és un dels mes importants patògens vegetals i agressius en l'agricultura i la silvicultura. La detecció primerenca i la identificació de les seves vies resulten de gran importància per a minimitzar l'amenaça que representen per als ecosistemes naturals. S'han desenvolupat i aplicat diferents mètodes moleculars per a la detecció de patògens de plantes en mostres ambientals. Aquests mètodes permeten una detecció i identificació de patògens ràpida i precisa fins i tot quan la quantitat d'inòcul és baixa. Per tant, es proposa un nou mètode millorat per a la seva detecció en mostres ambientals a partir de l'extracció d'ADN ambiental (eDNA) de diferents fonts (sòl, arrels i aigua) i diferents ecosistemes. L'objectiu del primer capítol va ser aplicar HTS (High Throughput Sequencing) per investigar la presència de Phytophthora en diferents comunitats de plantes en boscos naturals, plantacions i ambients aquàtics al nord d'Espanya. L'eDNA es va extraure del sòl i de l'aigua dels rius i rierols dels boscos de Fagus sylvatica i Abies alba i de plantacions de Chamaecyparis lawsoniana i Pseudotsuga menziesii al nord d'Espanya (bosc d'Irati i Villanúa). Es va dissenyar i va aplicar un assaig específic per a la detecció de Phytophthora mitjançant la seqüenciació massiva de amplicons basat en la regió ITS1. Diferents valors de threshold es van analitzar per a la separació òptima d'espècies de Phytophthora en les anàlisis bioinformàtics. L'agrupament al 99% va ser el millor criteri per separar la major part de les espècies de Phytophthora. Múltiples Unitats Operacionals Taxonòmiques Moleculars (MOTU) corresponents a 36 espècies diferents de Phytophthora es van amplificar en les mostres ambientals. La piroseqüenciació d'amplicons de mostres de sòl va revelar una diversitat baixa de Phytophthora (13 espècies) en comparació amb les 35 espècies detectades en mostres d'aigua. Tretze dels MOTU detectats en els rius i rierols no van mostrar homologia amb seqüències dipositades en les bases de dades, el que revela que la pirosequenciació de l'ADN ambiental és una estratègia útil per avaluar la diversitat d'espècies de Phytophthora en els ecosistemes naturals. Una vegada que la tècnica va ser desenvolupada i validada, es va proposar un altre objectiu enfocat en el decaïment de la carrasca. La carrasca (Quercus ilex) és l'espècie arbòria més representativa de la Península Ibèrica i l'arbre principal de les deveses. El decaïment de la carrasca en sòls no calcaris al sud-oest d'Espanya s'ha associat amb Phytophthora cinnamomi durant dècades. No obstant això, altres espècies de Phytophthora com P. quercina i P. psychrophila s'han associat amb el declivi de Quercus a la part oriental d'Espanya on predominen els sòls calcaris. Amb l'objectiu d'investigar la implicació de Phytophthora spp. en el declivi de la carrasca a l'est d'Espanya, es van seleccionar dos boscos en diferents zones geogràfiques (Alcoi i Vallivana) com a llocs de mostreig. Les mostres de sòl i arrel es van analitzar per piroseqüenciació d'amplicons. Els resultats de la seqüenciació massiva van mostrar la diversitat d'espècies de Phytophthora, i va revelar que un taxó mai aïllat de Phytophthora, anomenat de forma provisional Phytophthora taxon ballota, va ser l'espècie predominant en les dues àrees. A més, es va desenvolupar un assaig de PCR a temps real, basat en els resultats de la piroseqüenciació, per a la detecció d'aquest taxó de Phytophthora mai aïllat, i també per a la detecció de P. quercina. Els assajos de qPCR es van aplicar en mo
Catalá García, S. (2017). Development of DNA massive sequencing techniques and Real-Time PCR for the detection, identification and quantitation of Phytophthora spp. in environmental samples [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90644
TESIS
Books on the topic "Real-Time PCR technique"
1
Quantitative real-time PCR in applied microbiology. Norfolk, UK: Caister Academic Press, 2012.
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2
(Editor), W. Dietmaier, C. Wittwer (Editor), and N. Sivasubramanian (Editor), eds. Rapid Cycle Real Time PCR - Methods and Applications. Springer, 2002.
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1963-, Dietmaier W., Wittwer C. 1955-, and Sivasubramanian N, eds. Rapid cycle real-time PCR: Methods and applications : genetics and oncology. Berlin: Springer, 2002.
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Dyer, Paul S., Carol A. Munro, and Rosie E. Bradshaw. Fungal genetics. Edited by Christopher C. Kibbler, Richard Barton, Neil A. R. Gow, Susan Howell, Donna M. MacCallum, and Rohini J. Manuel. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780198755388.003.0005.
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Fungi have been long used as model organisms to investigate genetic and cellular processes. An overview is provided of how fungi function at a genetic level, including ploidy, gene structure, and gene flow by sexual and asexual processes. The tools used to study fungal genetics are then described, such techniques having widespread applications in medical mycology research. Classical genetic analysis includes the use of gene mapping by sexual crossing and tetrad analysis, and forward genetic experimentation based on mutagenesis, for which various mutant screening approaches are described. Molecular genetic analysis includes gene manipulation by transformation; different methods for gene knockout and targeting, and their application for forward and reverse genetic approaches, are outlined. Finally, molecular genetic methods used to study gene expression and function are reviewed, including use of inducible or constitutive overexpression, real-time PCR, cellular localization of gene products by fluorescent tagging, and detection of protein–protein interactions.
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United States. National Aeronautics and Space Administration., ed. Study of one- and two-dimensional filtering and deconvolution algorithms for a streaming array computer: Final report, appendix 5. [Washington, D.C: National Aeronautics and Space Administration, 1985.
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Study of one- and two-dimensional filtering and deconvolution algorithms for a streaming array computer: Final report : [appendices]. [Washington, D.C: National Aeronautics and Space Administration, 1985.
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Study of one- and two-dimensional filtering and deconvolution algorithms for a streaming array computer: Final report. [Washington, D.C: National Aeronautics and Space Administration, 1985.
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United States. National Aeronautics and Space Administration., ed. Study of one- and two-dimensional filtering and deconvolution algorithms for a streaming array computer: Final report. [Washington, D.C: National Aeronautics and Space Administration, 1985.
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United States. National Aeronautics and Space Administration., ed. Study of one- and two-dimensional filtering and deconvolution algorithms for a streaming array computer: Final report, appendix 5. [Washington, D.C: National Aeronautics and Space Administration, 1985.
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United States. National Aeronautics and Space Administration., ed. Study of one- and two-dimensional filtering and deconvolution algorithms for a streaming array computer: Final report : [appendices]. [Washington, D.C: National Aeronautics and Space Administration, 1985.
Find full textBook chapters on the topic "Real-Time PCR technique"
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Broll, Hermann. "Quantitative Real-Time PCR." In Molecular Biological and Immunological Techniques and Applications for Food Chemists, 59–83. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010. http://dx.doi.org/10.1002/9780470637685.ch3.
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Hawkins, Steve F. C., and Paul C. Guest. "Multiplex Analyses Using Real-Time Quantitative PCR." In Multiplex Biomarker Techniques, 125–33. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-6730-8_8.
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McAdam, Alexander J. "Real-Time and Digital PCR for Nucleic Acid Quantification." In Advanced Techniques in Diagnostic Microbiology, 377–87. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-33900-9_18.
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Yanase, Sumino, Hiroyoshi Sasahara, Momoko Nabetani, Kensuke Yamazawa, Keisuke Aoyagi, Akiko Mita, Yuichi Honma, and Yasuhiko Chiba. "Quantitative Real-Time RT-PCR Systems to Detect SARS-CoV-2." In Multiplex Biomarker Techniques, 89–97. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2395-4_7.
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Taliefar, M., S. Bradburn, G. Podda, and C. Murgatroyd. "Reverse Transcription Real-Time PCR Protocol for Gene Expression Analyses." In Handbook of Vascular Biology Techniques, 363–72. Dordrecht: Springer Netherlands, 2015. http://dx.doi.org/10.1007/978-94-017-9716-0_28.
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Gao, Jinling, Nesredin Kedir, Cody Kirk, Julio Hernandez, Xuedong Zhai, Junyu Wang, Tyler Tallman, Kamel Fezzaa, and Weinong Chen. "Real-Time Visualization of Damage Progression Inside GFRCs via High-Speed X-Ray PCI Technique." In Fracture, Fatigue, Failure and Damage Evolution , Volume 3, 69–72. Cham: Springer International Publishing, 2021. http://dx.doi.org/10.1007/978-3-030-60959-7_11.
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Nazarbakhsh, Behzad, and Azizah Abd Manaf. "Image Pre-processing Techniques for Enhancing the Performance of Real-Time Face Recognition System Using PCA." In Bio-inspiring Cyber Security and Cloud Services: Trends and Innovations, 383–422. Berlin, Heidelberg: Springer Berlin Heidelberg, 2014. http://dx.doi.org/10.1007/978-3-662-43616-5_15.
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Gentilal, Nichal, Ricardo Salvador, and Pedro Cavaleiro Miranda. "A Thermal Study of Tumor-Treating Fields for Glioblastoma Therapy." In Brain and Human Body Modeling 2020, 37–62. Cham: Springer International Publishing, 2020. http://dx.doi.org/10.1007/978-3-030-45623-8_3.
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AbstractTumor-treating fields (TTFields) is an antimitotic cancer treatment technique used for glioblastoma multiforme (GBM) and malignant pleural mesothelioma. Although the frequency used is not as high as in hyperthermia, temperature increases due to the Joule effect might be meaningful given the necessary time that these fields should be applied for. Post hoc analysis of the EF-11 clinical trial showed higher median overall survival in patients whose compliance was at least 18 h per day. To quantify these temperature increases and predict the thermal impact of TTFields delivery to the head, we used a realistic model created from MR images segmented in five tissues: scalp, skull, CSF, gray matter (GM), and white matter (WM). Through COMSOL Multiphysics, we solved Laplace’s equation for the electric field and Pennes’ equation for the temperature distribution. To mimic the therapy as realistically as possible, we also considered complete current shutdown whenever any transducer reached 41 °C to allow transducers and tissues’ temperature to decrease. Our results indicate an intermittent operation of Optune due to this necessary current shutdown. Localized temperature increases were seen, especially underneath the regions where the transducers were placed. Maximum temperature values were around 41.5 °C on the scalp and 38 °C on the brain. According to the literature, significant thermal impact is only predicted for the brain where the rise in temperature may lead to an increased BBB permeability and variation in the blood flow and neurotransmitter concentration. Additionally, our results showed that if the injected current is reduced by around 25% compared to Optune’s standard way of operating, then uninterrupted treatment might be attainable. These predictions might be used to improve TTFields delivery in real patients and to increase awareness regarding possible thermal effects not yet reported elsewhere.
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Villarreal Camacho, Jose Luis, and Alfonso Carlos Bettin Martinez. "Standardization of the PCR technique for the detection of bacterial pathogenic microorganisms inwater using as a model Salmonella spp. and E. coli." In Real-time PCR applied to bacterial waterborne pathogens detection and quantification, 77–88. Ediciones Universidad Simón Bolívar, 2017. http://dx.doi.org/10.17081/bonga.2616.c6.
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Khate, Kevisino, and Arambam Neelima. "Detection of COVID-19 Infection Using Chest X-Ray Images." In Machine Learning and AI Techniques in Interactive Medical Image Analysis, 83–105. IGI Global, 2022. http://dx.doi.org/10.4018/978-1-6684-4671-3.ch005.
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Coronavirus (COVID-19) is an infectious viral illness that causes health concerns. It was initially recorded in Wuhan (China). Early diagnosis of the disease aids in preventing its spread. Real-time reverse transcription-polymerase chain reaction (RT-PCR) is a laboratory test method for detecting the COVID-19 virus. To avoid the spread of COVID-19 disease, the researcher researched other techniques of diagnosis. One such technique is the classification of COVID-19 using medical images, notably chest x-ray (x-ray), computed tomography (CT), and ultrasound images. This chapter suggests merging canny edge detection techniques with traditional machine learning and deep learning techniques to diagnose COVID-19.
Conference papers on the topic "Real-Time PCR technique"
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Taha, Aza, Katan Ali, and Furat Sabeer. "Quantitation assay of hepatitis C virus RNA using real-time PCR technique." In 4th International Scientific Conference of Cihan University-Erbil on Biological Sciences. Cihan University-Erbil, 2017. http://dx.doi.org/10.24086/bios17.22.
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Fung, Tracy Helen, Shih-Hui Chao, Joseph E. Peach, and Deirdre R. Meldrum. "Liquid Crystal Thermography of an On-Chip Polymerase Chain Reaction Micro-Thermocycler." In ASME 4th International Conference on Nanochannels, Microchannels, and Minichannels. ASMEDC, 2006. http://dx.doi.org/10.1115/icnmm2006-96175.
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Microscale liquid crystal thermography is a technique to measure temperature distribution of microfabricated devices in real-time. This method utilizes a microscope to image the color map of a layer of temperature-sensitive encapsulated thermochromic liquid crystals (TLC) coated on a microfabricated device. This paper describes the TLC coating process on microscale devices, the characteristics of colorimetric hysteresis, and the calibration of temperature measurements. The calibrated measurements have been applied for characterization of an on-chip polymerase chain reaction (PCR) microscale thermocycler where precise and dynamic temperature control is essential for efficient DNA amplification. Tests on the micro-thermocycler were done around the ranges centered at 30 °C and 95 °C. The results illustrate the effects on the temperature distribution due to micro-thermocycler geometry, and provide important insight for micro-thermocycler design.
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Misu, Toshie, Yasutaka Matsuo, Shunsuke Iwamura, and Shinichi Sakaida. "Real-time implementation of UHDTV video coding system with super-resolution techniques." In 2013 Picture Coding Symposium (PCS). IEEE, 2013. http://dx.doi.org/10.1109/pcs.2013.6737713.
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Longhurst, Daniel V., and Donald Longhurst. "Infrared Monitoring Techniques for Real-Time Monitoring of Rotary Ball Mills." In 2009 IEEE-IAS/PCA Cement Industry Technical Conference Record. IEEE, 2009. http://dx.doi.org/10.1109/citcon.2009.5116174.
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Oh, Miae, Minhye Cho, Jae-Jin Choi, and Heekyung Park. "Abstract 3067: Improved detection of BRAF mutation in clinical samples using PNA-mediated real-time PCR clamping techniques." In Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL. American Association for Cancer Research, 2011. http://dx.doi.org/10.1158/1538-7445.am2011-3067.
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Torres, Melitsa J., Jose D. Posada, Jaime R. Garcia, and Marco E. Sanjuan. "Real-Time Fault Detection Application for Natural Gas Pipelines." In ASME 2012 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/imece2012-88927.
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The implementation of fault detection techniques in industrial systems for process monitoring has proven to be a useful tool to process operators supervising the plant’s operation conditions. As plants become more instrumented, more data is available for fault detection applications, if they are capable of demonstrate anticipation and low false alarm rates. A regional Natural Gas transportation system deals with these types of drawbacks. While the improvements are carried out, some effort should be done in order to improve the safety in operations. In this paper a data-driven technique was used to detect fault conditions along the pipeline, sectioning it in five partitions to increase the detection sensibility. To overcome the lack of quality in data, simulation software intended to gas controllers training and pipeline operation was used to simulate leaks scenarios. Some historic data with high quality is also used to create normal operation condition models by means of Principal Component Analysis. All simulated faults were detected in a reduced time gap and recent events related to third-party actions showed the tool proficiency to detecting faults in real time. In addition, it considers a fault normalized index per section indicating the fault persistence and aggressiveness in a single plot.
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Proctor, Frederick M., and Justin R. Hibbits. "Performance Comparisons of Timing Techniques in a Non-Real-Time Environment." In ASME 2005 International Design Engineering Technical Conferences and Computers and Information in Engineering Conference. ASMEDC, 2005. http://dx.doi.org/10.1115/detc2005-85234.
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General-purpose computers are increasingly being used for serious control applications, due to their prevalence, low cost and high performance. Real-time operating systems are available for PCs that overcome the nondeterminism inherent in desktop operating systems. Depending on the timing requirements, however, many users can get by with a non-real-time operating system. This paper discusses timing techniques applicable to non-real-time operating systems, using Linux as an example, and compares them with the performance that can be obtained with true real-time OSes.
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Cho, Minhye, Jae-Jin Choi, and Heekyung Park. "Abstract 5059: Detection of K-RAS mutation in clinical samples by one-step PNA-mediated real-time PCR clamping techniques." In Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC. American Association for Cancer Research, 2010. http://dx.doi.org/10.1158/1538-7445.am10-5059.
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Roy, Vishnu, Anurag Pandey, Amit Saxena, and Shivanjali Sharma. "Assessment of Machine Learning Techniques for Real-Time Prediction of Equivalent Circulating Density." In Offshore Technology Conference Asia. OTC, 2022. http://dx.doi.org/10.4043/31523-ms.
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Abstract The equivalent circulating density (ECD) is crucial in avoiding fluid losses or kicks while drilling. It's more critical in wells where the pore pressure gradient is close to the fracture pressure gradient. The conservation of mass and momentum determine the ECD, but this method does not account for other factors like torque, rotating speed, weight on bit, etc. These may affect the ECD directly or indirectly. The aim of this study is a practicality to predict the ECD using various machine learning techniques and to determine their effectiveness. The complete drilling dataset of an oil well from Texas was acquired. Over 16000 data points were obtained after the removal of the null values. The data was prepared by scaling it and conducting principal component analysis (PCA). PCA reduced the dimensionality of the dataset while retaining the information. Following this, five different machine learning regression techniques were used to predict the equivalent circulation density, namely, XGBoost, Random Forest, Support Vector Machine, Decision Tree, and Elastic net regression. The performance of these techniques was judged by comparing their R2 scores, mean squared errors (MSE), and root mean squared errors (RMSE). The results showed that ECD prediction through all the above machine learning techniques is a vital reality. Random forest regression emerged superior from the different methods used, illustrating the highest R2 score and the lowest MSE and RMSE. Its R2 for our model was 0.992, which is an excellent fit. It was followed by SVM, which had the second-lowest RMSE and an R2 of 0.987, close to the random forest technique. Elastic Net, Decision tree, and XG Boost in the respective order were at the bottom of the pool. Machine learning is a powerful tool at our disposal to effectively predict quantities in real-time that directly or indirectly depend on several parameters. It can even be effective when no direct correlation between the quantities is known. Thus, machine learning can significantly enhance our ability to optimize drilling operations by having quicker and more accurate predictions. The work shown in this study, if implemented, can provide the crew more time to respond to situations such as the occurrence of kicks and thus will lead to safer operations.
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Mousavi, Seyed Reza, and Abbas Samani. "Realtime Ultrasound Prostate Young’s Modulus Reconstruction Technique Using a Full Inversion Approach." In ASME 2010 International Mechanical Engineering Congress and Exposition. ASMEDC, 2010. http://dx.doi.org/10.1115/imece2010-37747.
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In this work, we present a real-time prostate elastography reconstruction technique which incorporates an accelerated method of tissue stress calculation in its algorithms. The accelerated FE method uses a database of prostate-tumor configurations obtained from imaging. These configurations undergoing specific mechanical loading, e.g. US probe, are modeled and analyzed using conventional FEM to obtain the corresponding stress fields. Principal component analysis (PCA) is used to obtain the main modes of shape and stress fields. As such, the shape and stress fields can be described by these main modes weighted by a small number of weight factors. Next, an efficient mapping technique is developed to relate the weight factors of shape to those of the stress fields. We used Artificial Neural Network (ANN) and PCA based regression for this mapping. Once the mapping function is obtained it can be used for analyzing prostate shapes not included in the database. For a typical prostate, our results indicate that analysis using our technique takes less than 0.1 seconds on a desktop computer irrespective of the model size, while the maximum stress error is less than 5% per element. This mapping is then used for our novel real-time Young’s modulus reconstruction technique in which prostate and tumor moduli are updated iteratively using strain images acquired from an ultrasound imaging system and stress field estimated with the proposed method. Results of elastography show that relative Young’s moduli can be reconstructed fairly accurately.
Reports on the topic "Real-Time PCR technique"
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Mohammadian, Abolfazl, Amir Bahador Parsa, Homa Taghipour, Amir Davatgari, and Motahare Mohammadi. Best Practice Operation of Reversible Express Lanes for the Kennedy Expressway. Illinois Center for Transportation, September 2021. http://dx.doi.org/10.36501/0197-9191/21-033.
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Reversible lanes in Chicago’s Kennedy Expressway are an available infrastructure that can significantly improve traffic performance; however, a special focus on congestion management is required to improve their operation. This research project aims to evaluate and improve the operation of reversible lanes in the Kennedy Expressway. The Kennedy Expressway is a nearly 18-mile-long freeway in Chicago, Illinois, that connects in the southeast to northwest direction between the West Loop and O’Hare International Airport. There are two approximately 8-mile reversible lanes in the Kennedy Expressway’s median, where I-94 merges into I-90, and there are three entrance gates in each direction of this corridor. The purpose of the reversible lanes is to help the congested direction of the Kennedy Expressway increase its traffic flow and decrease the delay in the whole corridor. Currently, experts in a control location switch the direction of the reversible lanes two to three times per day by observing real-time traffic conditions captured by a traffic surveillance camera. In general, inbound gates are opened and outbound gates are closed around midnight because morning traffic is usually heavier toward the central city neighborhoods. In contrast, evening peak-hour traffic is usually heavier toward the outbound direction, so the direction of the reversible lanes is switched from inbound to outbound around noon. This study evaluates the Kennedy Expressway’s current reversing operation. Different indices are generated for the corridor to measure the reversible lanes’ performance, and a data-driven approach is selected to find the best time to start the operation. Subsequently, real-time and offline instruction for the operation of the reversible lanes is provided through employing deep learning and statistical techniques. In addition, an offline timetable is also provided through an optimization technique. Eventually, integration of the data-driven and optimization techniques results in the best practice operation of the reversible lanes.
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Heifetz, Yael, and Michael Bender. Success and failure in insect fertilization and reproduction - the role of the female accessory glands. United States Department of Agriculture, December 2006. http://dx.doi.org/10.32747/2006.7695586.bard.
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The research problem. Understanding of insect reproduction has been critical to the design of insect pest control strategies including disruptions of mate-finding, courtship and sperm transfer by male insects. It is well known that males transfer proteins to females during mating that profoundly affect female reproductive physiology, but little is known about the molecular basis of female mating response and no attempts have yet been made to interfere with female post-mating responses that directly bear on the efficacy of fertilization. The female reproductive tract provides a crucial environment for the events of fertilization yet thus far those events and the role of the female tract in influencing them are poorly understood. For this project, we have chosen to focus on the lower reproductive tract because it is the site of two processes critical to reproduction: sperm management (storage, maintenance, and release from storage) and fertilization. E,fforts during this project period centered on the elucidation of mating responses in the female lower reproductive tract The central goals of this project were: 1. To identify mating-responsive genes in the female lower reproductive tract using DNA microarray technology. 2. In parallel, to identify mating-responsive genes in these tissues using proteomic assays (2D gels and LC-MS/MS techniques). 3. To integrate proteomic and genomic analyses of reproductive tract gene expression to identify significant genes for functional analysis. Our main achievements were: 1. Identification of mating-responsive genes in the female lower reproductive tract. We identified 539 mating-responsive genes using genomic and proteomic approaches. This analysis revealed a shift from gene silencing to gene activation soon after mating and a peak in differential gene expression at 6 hours post-mating. In addition, comparison of the two datasets revealed an expression pattern consistent with the model that important reproductive proteins are pre-programmed for synthesis prior to mating. This work was published in Mack et al. (2006). Validation experiments using real-time PCR techniques suggest that microarray assays provide a conservativestimate of the true transcriptional activity in reproductive tissues. 2.lntegration of proteomics and genomics data sets. We compared the expression profiles from DNA microarray data with the proteins identified in our proteomic experiments. Although comparing the two data sets poses analyical challenges, it provides a more complete view of gene expression as well as insights into how specific genes may be regulated. This work was published in Mack et al. (2006). 3. Development of primary reproductive tract cell cultures. We developed primary cell cultures of dispersed reproductive tract cell types and determined conditions for organ culture of the entire reproductive tract. This work will allow us to rapidly screen mating-responsive genes for a variety of reproductive-tract specifi c functions. Scientific and agricultural significance. Together, these studies have defined the genetic response to mating in a part of the female reproductive tract that is critical for successful fertllization and have identified alarge set of mating-responsive genes. This work is the first to combine both genomic and proteomic approaches in determining female mating response in these tissues and has provided important insights into insect reproductive behavior.