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RAPID DNA-BASED POINT-OF-CARE DIAGNOSTICS

YERI, ASHISH S (2012) RAPID DNA-BASED POINT-OF-CARE DIAGNOSTICS. Doctoral Dissertation, University of Pittsburgh.

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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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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmail
    Committee ChairGAO, DIgaod@pitt.edu
    Committee MemberVATS, ABHAYabhay.vats@chp.edu
    Committee MemberLITTLE, STEVEN Rsrlittle@pitt.edu
    Committee MemberATAAI, MOHAMMAD Mataai@pitt.edu
    Title: RAPID DNA-BASED POINT-OF-CARE DIAGNOSTICS
    Status: Published
    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.
    Date: 02 February 2012
    Date Type: Publication
    Defense Date: 23 November 2011
    Approval Date: 02 February 2012
    Submission Date: 29 November 2011
    Release Date: 02 February 2012
    Access Restriction: No restriction; Release the ETD for access worldwide immediately.
    Patent pending: No
    Number of Pages: 127
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    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
    Schools and Programs: Swanson School of Engineering > Chemical Engineering
    Date Deposited: 02 Feb 2012 11:01
    Last Modified: 01 Jul 2014 16:28

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