Noor, Nik Zuraina Nik Mohd
(2020)
Thermostabilized multiplex pcr assay for detection of selected bacteria associated with respiratory tract infections among malaysian hajj pilgrims.
PhD thesis, Universiti Sains Malaysia.
Abstract
Respiratory tract infections (RTIs) are the commonest health problem during
the annual Hajj pilrimage. Common bacteria associated with RTIs include Klebsiella
pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus
pneumoniae, Mycobacterium tuberculosis and Pseudomonas aeruginosa. Rapid
detection of these pathogens could facilitate towards effective therapies. Therefore,
this study aimed to develop and evaluate a thermostabilized polymerase chain reaction
(PCR) assay for simultaneous detection of these six bacteria. The first step involved
designing specific primers for the target bacteria and an internal amplification control
(IAC). Each set of primers was evaluated to analyze for their specificity and
sensitivity. A multiplex PCR was then developed by optimizing the concentration of
primers and other components. Initial accuracy of the multiplex PCR was determined
on clinical isolates. Subsequently, this assay had undergone lyophilization process in
the presence of trehalose as the sugar-stabilizer. The assay stability was tested at
different sets of temperature for different time-intervals. In the last stage, this assay
was evaluated on the sputum specimens from Hospital USM and further evaluated at
the field level using the specimens from Malaysian Hajj pilgrims. Results indicated
that all the designed primers were specific to the respective target bacteria. The
optimized concentrations of primers for bacteria (0.4 μM) and IAC (0.2 mM), MgCl2
(2.5 mM), dNTPs (0.2 mM) and Taq DNA polymerase enzyme (0.75 unit) were used
in the development of multiplex PCR assay. Initial evaluation on bacterial isolates
showed that the assay was 100% accurate on both target and non-target bacteria (n =145) (analytical specificity) with the lowest limit of detection was 10 pg DNA (200
bacterial cell) (analytical sensitivity). Lyophilization of this assay was successfully
carried out in the presence of 6% trehalose in the PCR reagent. The assay was stable
at the ambient temperature (25ºC) for at least six months. The sensitivity, specificity
and accuracy of this assay were 100%, 92% and 95%, respectively on cinical sputum
specimens (n = 200). Field evaluation on specimens from Malaysian Hajj pilgrims
ensued the sensitivity and specificity of 100% and 92%, respectively, with the
accuracy of 97%. From this study, two main bacteria detected from the clinical and
Hajj sputum specimens were K. pneumoniae and H. influenzae, respectively. In
conclusion, the rapidity, convenience, thermal-stable and reliable, could enable the
application of this thermostabilized multiplex PCR assay to be used as a molecular
diagnostic tool for the detection of six respiratory bacteria.
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