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Thesis
Development of a real-time PCR assay for detection of hepatitis B virus 1762T/1764A double mutation for the screening of Hepatocellular Carcinoma
Master of Science (M.S.), Drexel University
May 2009
DOI:
https://doi.org/10.17918/etd-3011
Abstract
The 1762T/1764A double mutation of the hepatitis B virus (HBV) located in the basal core promoter of the HBV genome, has been suggested to be a potential biomarker for hepatocellular carcinoma (HCC) among individuals with chronic HBV infection. Previously, we have demonstrated that urine contains fragmented, low molecular weight (300 bp in size) DNA from the body circulation of fluids and can be used for cancer detection. The ultimate goal of this experimental investigation is to explore the potential to develop an assay for the detection of this HBV double mutation associated with HCC with future works in the area of applying this assay for urine testing. Here, we developed an ultra-sensitive real-time PCR assay suitable for utilizing low molecular weight substrate by using hybridization probes and an oligonucleotide clamp containing locked nucleic acids (LNAs). Hybridization probes provide melting curve analysis capabilities which eliminate post-PCR manipulation and minimize the human error handling to a ofe-step process. Labor and time was minimized to less than two hours per assay run. The LNA-containing oligonucleotide clamp was specific for the wild type HBV and demonstrated the ability to suppress the amplification of the wild type HBV templates and inhibit the binding of the WT templates to the fluorescence probes. As a result, the detection of HBV double mutation was made possible in the presence of 3000-fold excess of the wild type genome through the use of post-PCR melting curve analysis capabilities; an improvement from the detection of 20 fold excess wild type genome. Thus, this assay can potentially be used in a real-time PCR based urine test for HCC screening. Overall, PCR amplification coupled with the melting curve analysis generated a quick, simple, and highly sensitive tool for the detection of this HBV double mutation.
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Details
- Title
- Development of a real-time PCR assay for detection of hepatitis B virus 1762T/1764A double mutation for the screening of Hepatocellular Carcinoma
- Creators
- Selena Lin - DU
- Contributors
- Ying-Hsiu Su (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- School of Biomedical Engineering, Science, and Health Systems (1997-2026); Drexel University
- Other Identifier
- 3011; 991014632294004721