Optical methods for the detection of Neisseria meningitidis-specific DNA

Thoduka, S 2009, Optical methods for the detection of Neisseria meningitidis-specific DNA, Doctor of Philosophy (PhD), Applied Sciences, RMIT University.


Document type: Thesis
Collection: Theses

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Title Optical methods for the detection of Neisseria meningitidis-specific DNA
Author(s) Thoduka, S
Year 2009
Abstract Meningococcal disease, whose causative agent is Neisseria meningitides, has a mortality rate of 100% if untreated and remains 7-10% even if treated with a morbidity of about 20%. The disease may result in death within 24 hours of the first symptoms. Hence, rapid diagnosis and identification of N. meningitidis in clinical samples is essential for the management of the disease and the administration of appropriate and effective treatment. Current methods for detection have limitations in sensitivity or time required. The main aim of this thesis was to evaluate two methods, which use the unique properties of gold nanoparticles, as rapid and simple detection methods for the identification of N. meningitidis-specific DNA sequences.

A N. meningitidis-specific DNA sequence (porA4) was selected from the porA gene that is only expressed in N. meningitidis. The probes designed against this sequence were conjugated to gold nanoparticles using conditions optimised in this study and were then employed in the two detection methods.

The first detection method utilised the changes in the plasmon resonance of gold nanoparticles produced by the target DNA-directed aggregation of nanoparticles and could be seen by a change in colour of the solution. Conditions optimised for hybridisation of DNA to nanoparticle-bound probes were used to achieve the greatest plasmon resonance or colour shift. Meningococcal genomic DNA could not be detected by this method. However the porA gene PCR product was detected within 1 hour when a minimum of 2 nmole of the target DNA was present. The PCR required at least 103 copies of template genomic DNA to amplify the target gene.

The second detection method utilised DNA probes labelled with fluorophores and monitored the hybridisation of target DNA to the probes by associated changes in fluorescence. In these schemes, the fluorophore label was quenched by the gold nanoparticles in the absence of target DNA however, when target DNA was present, the DNA probes hybridised to it and the fluorescence of the label could be detected. Both time-resolved and steady-state fluorescence measurements were used for the detection of meningococcal DNA. Two different fluorophore-labelled probes used in this study could detect the presence of synthetic target DNA as well as meningococcal DNA in the order of 0.1 pmole. The sensitivity of this method was greater compared with the plasmon resonance shift method and required a shorter time for detection.

The concentration of meningococcal cells in blood and cerebrospinal fluid can vary from 0 to 107 cfu/mL therefore, any method that is used in the diagnosis of meningococcal disease would require sensitivity to detect low numbers of organisms in clinical samples. In order for either of the detection methods used in this study to be clinically applicable, the samples need to undergo a form of DNA amplification such as PCR to enhance the sensitivity prior to detection. The sensitivity, specificity and speed of these detection methods suggest that the use of DNA-gold nanoparticle conjugates can have a wide range of applications, both for the detection of other pathogens as well as for genetic screening.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Sciences
Keyword(s) Neisseria meningitidis
meningococcal disease
gold nanoparticles
plasmon resonance
fluorescence
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Created: Wed, 27 Jun 2012, 10:10:51 EST by Guy Aron
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