Ali, Mohammad Ridhuan Mohd
(2018)
Development of thermostable multiplex qPCR for simultaneous detection of pathogens associated with febrile illnesses.
Masters thesis, Universiti Sains Malaysia.
Abstract
To date, several molecular assays are available for the diagnosis of
leptospirosis, melioidosis, invasive salmonellosis and malaria. These reported assays
allow early detection of pathogens nucleic acids; hence serve as potential alternatives
to the conventional methods including culture, serology and microscopic
examination. However, majority of the reported molecular assays lack of internal
amplification control (IAC), do not adhere to the MIQE guidelines and usually detect
a single organism. To address these major limitations of current molecular assays,
this study is aimed to develop a multiplex TaqMan hydrolysis probe-based qPCR
that can detect nucleic acids of four different organisms (Leptospira, B.
pseudomallei, Salmonella and Plasmodium) and an IAC within a single reaction. To
achieve these objectives, four pairs of primers and four TaqMan hydrolysis probes
were designed against Leptospira, B. pseudomallei, Salmonella and Plasmodium
genomes. These primer pairs were conjugated with a universal adapter (UN-adapter)
and combined with UN primers in order to increase the performance of the assay. In
addition, another pair of primers and probe against Mycobacterium tuberculosis rpoB
gene and Entamoeba histolytica HLY5mc1 gene was also developed as IAC tool.
Validation of the assay was performed according to the MIQE guidelines. First,
analytical sensitivity of the multiplex qPCR assay was evaluated on 10-fold serial
dilutions of genomic DNA from each microbial target, as well as its specificity on
357 microbial isolates, made up of 131 Leptospira isolates, 105 B. pseudomallei
isolates, 44 Salmonella isolates, 31 Plasmodium strains and 46 other organisms.
Following stability testing at different temperatures, the clinical performance of the
qPCR assay was evaluated on 518 retrospective specimens from suspected patients.
In this study, it was found that the developed multiplex qPCR assay correctly
amplified and differentiated all Leptospira, B. pseudomallei, Salmonella and
Plasmodium isolates, with limit of detections (LODs) of 5.61 copies, 8.24 copies,
19.3 copies and 18.1 copies per reaction, respectively. No undesired amplification
was observed in other tested organisms. Similarly, the clinical evaluation showed
that the qPCR assays had clinical sensitivities of 100% and clinical specificities of
between 98.8% and 100%. No evidence of PCR inhibition was observed. In terms of
stability, in the presence of 5% trehalose, the lyophilised qPCR mix had an estimated
shelf-life of 84.7 days, at ambient temperature. Overall, this study successfully
developed a multiplex qPCR assay for early detection of four common febrile
causing infections, in particular leptospirosis, melioidosis, invasive salmonellosis and
malaria, at high sensitivity and specificity, both analytically and clinically.
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