Kaye, Steven; (1997) The application of the polymerase chain reaction to the study of human immunodeficiency virus type 1 infection. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The polymerase chain reaction (PCR) amplifies genomic DNA sequences in an in vitro reaction, allowing detection, quantification and characterisation of the amplified sequence. This thesis describes the application of PCR to the investigation of human immunodeficiency virus type 1 (HIV-1) infection. The aim has been to develop methods which can be applied directly to clinical samples taken from infected individuals and which do not require the in vitro culture of the virus. Methods for the preparation and storage of suitable samples for amplification by PCR have been developed and evaluated including a whole blood cryopreservation method Sequence specific oligonucleotides have been designed for the diagnostic detection of HIV-1 in clinical samples by PCR. The diagnostic specificity and sensitivity of the method have been determined using characterised virus isolates and clinical samples collected from cohorts of infected adults and babies born to infected women. A PCR has been designed to amplify the reverse transcriptase (RT) gene of HIV-1 and the amplified product has been analysed with a novel quantitative point mutation assay (PMA). The PMA was designed to quantify the proportions of wild-type and mutant sequence at a given point in mixed virus sequences derived from clinical material, and was used to analyse mutations associated with resistance to antiretroviral drugs. PMA has been used to detect genomic drug resistance in a number of HIV-1 infected subject groups receiving zidovudine therapy, and the results analysed in parallel with other laboratory findings. The assay has been used to identify drug resistant virus in cerebrospinal fluid and to demonstrate the dynamics of viral replication in vivo. The rapid throughput of samples by PMA has led to it becoming a cornerstone of the virological analysis of MRC antiretroviral trials, being applied to both mono- and combination-therapy trials.

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