Introduction of internal controls to a Neisseria meningitidis realtime PCR assay
Cardiff Metropolitan University
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Bacterial meningitis is a major cause of morbidity and mortality in people of all ages. Neisseria meningitidis is one of the causative organisms of bacterial meningitis which mainly affects infants and young adults. At the beginning of the 1900's mortality rates were very high and even with modern medical techniques and treatments mortality rates remain around 10%. Fast recognition of bacterial meningitis and identification of the causative organism is of utmost importance for a favourable clinical outcome. Real-time PCR has enabled the detection time of organisms in clinical samples to be reduced significantly. It also does not rely on the organism being viable at the time of testing, which is particular importance when testing cerebral spinal fluid (CSF) and EDTA blood samples for N. meningitis. Quality control is a necessity of modern laboratory practice. The introduction of an internal control into the current N. meningitidis Real-time PCR assay was felt to be essential in order to better validate results and reduce reporting times. The detection of human ribonuclease P (RNase P) was used as an internal control. Experimentation found that it was not present in N. meningitidis cells, but was present in sufficient quantities to be detected in both blood and CSF samples. It was not found to interfere or cross react with the primers and probes of CtrA which were used to detect the presence of N. meningitidis. The use of a duplex assay for typing any positive samples was also investigated. This utilised SiaD B and C primers and probes. The optimal final concentrations for both were probes at a final concentration of 0.2µM and the primers at a final concentration of 0.1µM. As a result of these experiments a new and more robust Real-time PCR assay for the detection and typing of N. meningitidis DNA has been developed.
MSc Biomedical Sciences
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