Title:
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Development of DNA microarray system for the diagnosis of virus infections of the central nervous system
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The increasing numbers of neurovirulent viruses, their similar clinical
manifestations and the limited number of detectable viruses per assay increase
the urge for a more comprehensive and rapid diagnostic assay. This will be
reflected on patient management and community savings. Although PCR is a
powerful tool for nucleic acid amplification, its specific amplification confines
its use to a small number of viruses. Whole genome amplification using
random techniques may represent suitable comprehensive alternatives to PCR
when combined with the specific DNA microarray technologies.
In this project, variable parameters in the field of DNA microarray technology
were investigated such as probe design, surface chemistry and hybridisation
optimisation. Target preparation in terms of random amplification and labelling
was also investigated. The possibility of moving the assay from the expensive
fluorescent protocols to cheaper alternatives more suited to widespread use was
explored using manual colorimetric assays in place of automated fluorescent
detection procedures.
After extensive developmental work, a single protocol was proved to
differentiate between herpes simplex virus 1, herpes simplex 2, Epstein Barr
virus, cytomegalovirus, varicella zoster virus, human herpesvirus 6, and
coxsackievirus. The sensitivity of the assay was close to the sensitivity of
reference laboratories using PCR based techniques. The colorimetric assay also
provided a cost-effective sensitive format that is capable of differentiating
between various viruses. This assay offers an opportunity to develop a single process capable of simultaneous detection of a large number of possible central nervous viruses in a shorter period of time. The initial colorimetric results render the assay more applicable in laboratories with low technical facilities.
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