Analysis of the Mycoplasma hominis hsp70 gene and development of a PCR ELISA assay.
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Date
1998
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Abstract
Mycoplasmas conform most closely with the theoretical concept of 'minimum cells', existing as
the smallest, free-living organisms capable of self-replication. They survive as parasites of plants,
insects, animals or humans, with the most common human colonising species being Mycoplasma
hominis. M. hominis has been characterised as a human pathogen responsible for a variety of
infections, which pose a significant threat particularly to immunocompromised patients and
neonates. However little has been elucidated about the cell physiology and molecular structure
of this organism. Of interest to this study were the investigation of the heat shock response of
M. hominis and the diagnostic assays used for its detection.
The heat shock response is a ubiquitous physiological feature of all organisms and displays
unprecedented conservation. This phenomenon is particularly evident in the 70 kDa family of
heat shock proteins (hsp70) which exhibits a high degree of homology between different species.
The hsp70 gene from M. hominis was cloned and preliminary partial sequencing indicated the
similarity with other hsp70 homologs. The regulation of hsp70 expression at the transcriptional
and translational levels was investigated. The level of hsp70 mRNA was found to increase
correspondingly in response to heat shock, more visibly than the level of hsp70 protein.
However imrnunochemical studies of the M. hominis hsp70 translation product demonstrated
further the homology with other species.
To facilitate rapid diagnosis of M. hominis infections, a PCR ELISA diagnostic assay was
developed and optimised. The amplification of a conserved region of the M. hominis 16S rRNA
gene was linked to subsequent hybridisation to an appropriate capture probe in a microtiter plate
format. The sensitivity of the assay was comparable to other molecular assays although the PCR
ELISA produces more rapid results and is less labour intensive.
Description
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.
Keywords
Enzyme-Linked immunosorbent assay., Immuno enzyme technique., Mycoplasma hominis., Heatshock proteins., Mycoplasmatales., Microbiological--Assay., Theses--Biochemistry.