YERI, ASHISH S RAPID DNA-BASED POINT-OF-CARE DIAGNOSTICS. Doctoral Dissertation, University of Pittsburgh.
Abstract
DNA-based biosensors for detection of genetic mutations and infectious diseases have attracted a lot of attention recently owing to the large amount of genetic information available and the high specificity of these sensors due to the uniqueness of DNA sequences. In the first part of my work, the viscoelastic properties of single-strand DNA are made use of to rapidly screen for disease causing mutations. Single strand DNA (ssDNA) films have viscoelastic properties which are dependent on their 3-D conformation in solution which in turn is dependent on their base sequence. The mutation of the p53 gene which is responsible for almost half of all cancers was chosen as our case study. The appreciable differences in the viscoelastic properties between the p53 wild type ssDNA film and the p53 mutant R type ssDNA film were evaluated using a quartz crystal resonator showing that this method holds much promise to be used as a rapid DNA mutation screening technique. In the second part of my work, we have made use of novel isothermal DNA amplification techniques to specifically amplify the target DNA which is then detected on lateral flow strips in a low cost manner. Escherichia coli and Klebsiella pneumoniae are responsible for a large number of infections including Urinary Tract Infection (UTI) and life threatening conditions such as neonatal meningitis and neonatal sepsis. A rapid and sensitive detection scheme has been developed for these bacteria by amplification of the DNA with Loop mediated isothermal amplification technique (LAMP) and its subsequent detection on lateral flow strips. Patient urine samples were screened for the presence of these two organisms and the LAMP-lateral flow detection scheme is comparable to the bacterial culture methods which is the current gold standard. Furthermore, multiplexing the amplification and detection has been demonstrated successfully which shows great potential to be employed as a reliable point-of-care diagnostic tool in the clinical setting.
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Details |
| Item Type: | University of Pittsburgh ETD |
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| ETD Committee: | | ETD Committee Type | Committee Member | Email |
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| Committee Chair | GAO, DI | gaod@pitt.edu | | Committee Member | VATS, ABHAY | abhay.vats@chp.edu | | Committee Member | LITTLE, STEVEN R | srlittle@pitt.edu | | Committee Member | ATAAI, MOHAMMAD M | ataai@pitt.edu |
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| Title: | RAPID DNA-BASED POINT-OF-CARE DIAGNOSTICS |
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| Status: | Published |
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| Abstract: | DNA-based biosensors for detection of genetic mutations and infectious diseases have attracted a lot of attention recently owing to the large amount of genetic information available and the high specificity of these sensors due to the uniqueness of DNA sequences. In the first part of my work, the viscoelastic properties of single-strand DNA are made use of to rapidly screen for disease causing mutations. Single strand DNA (ssDNA) films have viscoelastic properties which are dependent on their 3-D conformation in solution which in turn is dependent on their base sequence. The mutation of the p53 gene which is responsible for almost half of all cancers was chosen as our case study. The appreciable differences in the viscoelastic properties between the p53 wild type ssDNA film and the p53 mutant R type ssDNA film were evaluated using a quartz crystal resonator showing that this method holds much promise to be used as a rapid DNA mutation screening technique. In the second part of my work, we have made use of novel isothermal DNA amplification techniques to specifically amplify the target DNA which is then detected on lateral flow strips in a low cost manner. Escherichia coli and Klebsiella pneumoniae are responsible for a large number of infections including Urinary Tract Infection (UTI) and life threatening conditions such as neonatal meningitis and neonatal sepsis. A rapid and sensitive detection scheme has been developed for these bacteria by amplification of the DNA with Loop mediated isothermal amplification technique (LAMP) and its subsequent detection on lateral flow strips. Patient urine samples were screened for the presence of these two organisms and the LAMP-lateral flow detection scheme is comparable to the bacterial culture methods which is the current gold standard. Furthermore, multiplexing the amplification and detection has been demonstrated successfully which shows great potential to be employed as a reliable point-of-care diagnostic tool in the clinical setting. |
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| Defense Date: | 23 November 2011 |
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| Approval Date: | 02 February 2012 |
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| Submission Date: | 29 November 2011 |
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| Release Date: | 02 February 2012 |
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| Access Restriction: | No restriction; Release the ETD for access worldwide immediately. |
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| Patent pending: | No |
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| Number of Pages: | 127 |
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| Institution: | University of Pittsburgh |
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| Thesis Type: | Doctoral Dissertation |
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| Refereed: | Yes |
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| Degree: | PhD - Doctor of Philosophy |
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| Uncontrolled Keywords: | DNA biosensors, mutation screening, viscoelastic modeling, voight, antibody immobilization, langmuir isotherm, sips isotherm, Loop mediated isothermal amplification, LAMP, lateral flow, E. coli, K. pneumoniae |
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| Schools and Programs: | Swanson School of Engineering > Chemical Engineering |
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| Date Deposited: | 02 Feb 2012 11:01 |
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| Last Modified: | 03 Feb 2012 01:15 |
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