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Title: The application of the polymerase chain reaction to the detection and characterization of human immunodeficiency virus and hepatitis B virus nucleic acid
Author: Nicholson, Wendy Jane
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1994
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This thesis considers the application of PCR to the detection and characterisation of human immunodeficiency virus (HIV) nucleic acid and hepatitis B virus (HBV) DNA in clinical samples. Primers were selected from the po1 and env sequences for the detection of HIV-1 DNA, and amplifications of the envelope gene were used to demonstrate sequence variability. HIV-1 DNA was detected in all 32 patients (59 of the 61 PBMC samples tested). Viral DNA sequences were detected in nuclear extracts tested, 71% of cytoplasmic extracts, and in 50% of purified monocytic cells. Primers were selected to differentiate between HIV-1 DNA structural forms. Covalently closed circular unintegrated viral DNA (CUVD) with 1 or 2 LTR sequences was detected by amplifying across the junction site, and linear unintegrated viral DNA (LUVD) was detected using a ligation mediated PCR (L-PCR). CUVD was detected in 65% of patient samples and was associated with disease progression (0.02>P>0.01). The detection of LUVD was negative for all patient samples. HBV DNA was detected in patient serum samples using 4 specific primers in a nested PCR. The primers were selected to hybridize to the S-gene sequence of HBV using the nucleotide consensus sequence for 5 HBsAg subtypes (adrcg, adw, adyw, ayw, and awy2). The presence of HBeAg in serum is indicative of infectivity. The detection of HBV DNA by PCR was compared with HBe status in 115 patient samples. All HBeAg positive and 25% of HBeAg negative samples were positive for HBV DNA. To determine the source of the viral DNA in these samples, the DNA extraction method was applied to free virus, and IgG and IgM complexed virus to establish the association of viral DNA with free virus and HBeAg.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available