Patou, Gary; (1992) Development and application of nested PCR for the detection of parvovirus B19 DNA. Doctoral thesis (M.D), UCL (University College London).

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Abstract

The development and characterisation of nested polymerase chain reaction (PCR) for the detection of human parvovirus B19 DNA is described. Two sets of oligonucleotide primers were used, one set have been described previously and a novel set were developed. The PCR reaction conditions were optimised for magnesium concentration, oligonucleotide primer concentration, extension time, reaction buffer and the serum treatment used for extraction of B19 DNA. The assay was characterised with a series of diagnostic sera validated by dot blot hybridisation for B19 DNA and by class specific capture radio-immunoassays for the detection of B19 IgM and IgG. The PCR assay was also applied to serial sera, throat swabs and peripheral mononuclear cells (PBMNC) collected during an outbreak of parvovirus B19. B19 DNA was detectable in the throat swabs and in the PBMNC fraction of whole blood. In situ hybridisation and immunolabelling were used in an attempt to determine the site of B19 DNA within the PBMNC fraction. B19 PCR was then applied to a number of clinical situations where the virus may play a role but where existing diagnostic tests are inadequate or require invasive procedures. PCR was used for the potential detection of B19 infection in immunocompromised patients in whom antibody responses are impaired and in anaemic malaria infected patients in whom hypergammaglobulinaemia may interfere with the detection of B19 specific antibodies. PCR was also used in the neonatal situation where little or no B19 IgM is produced and for the non-invasive diagnosis of fetal infection which currently requires fetal blood sampling. The PCR assay was also used to explore the role of B19 virus in patients with a variety of arthritic conditions. Finally, B19 PCR was applied to the detection of B19 DNA in clotting factor concentrates.

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