Development of thermostable multiplex qPCR for simultaneous detection of pathogens associated with febrile illnesses
Year: 2018
Type: dissertation
Abstract: To date, several molecular assays are available for the diagnosis of \nleptospirosis, melioidosis, invasive salmonellosis and malaria. These reported assays \nallow early detection of pathogens nucleic acids; hence serve as potential alternatives \nto the conventional methods including culture, serology and microscopic \nexamination. However, majority of the reported molecular assays lack of internal \namplification control (IAC), do not adhere to the MIQE guidelines and usually detect \na single organism. To address these major limitations of current molecular assays, \nthis study is aimed to develop a multiplex TaqMan hydrolysis probe-based qPCR \nthat can detect nucleic acids of four different organisms (Leptospira, B. \npseudomallei, Salmonella and Plasmodium) and an IAC within a single reaction. To \nachieve these objectives, four pairs of primers and four TaqMan hydrolysis probes \nwere designed against Leptospira, B. pseudomallei, Salmonella and Plasmodium \ngenomes. These primer pairs were conjugated with a universal adapter (UN-adapter) \nand combined with UN primers in order to increase the performance of the assay. In \naddition, another pair of primers and probe against Mycobacterium tuberculosis rpoB \ngene and Entamoeba histolytica HLY5mc1 gene was also developed as IAC tool. \nValidation of the assay was performed according to the MIQE guidelines. First, \nanalytical sensitivity of the multiplex qPCR assay was evaluated on 10-fold serial \ndilutions of genomic DNA from each microbial target, as well as its specificity on \n357 microbial isolates, made up of 131 Leptospira isolates, 105 B. pseudomallei \nisolates, 44 Salmonella isolates, 31 Plasmodium strains and 46 other organisms. \nFollowing stability testing at different temperatures, the clinical performance of the \nqPCR assay was evaluated on 518 retrospective specimens from suspected patients. \nIn this study, it was found that the developed multiplex qPCR assay correctly \namplified and differentiated all Leptospira, B. pseudomallei, Salmonella and \nPlasmodium isolates, with limit of detections (LODs) of 5.61 copies, 8.24 copies, \n19.3 copies and 18.1 copies per reaction, respectively. No undesired amplification \nwas observed in other tested organisms. Similarly, the clinical evaluation showed \nthat the qPCR assays had clinical sensitivities of 100% and clinical specificities of \nbetween 98.8% and 100%. No evidence of PCR inhibition was observed. In terms of \nstability, in the presence of 5% trehalose, the lyophilised qPCR mix had an estimated \nshelf-life of 84.7 days, at ambient temperature. Overall, this study successfully \ndeveloped a multiplex qPCR assay for early detection of four common febrile \ncausing infections, in particular leptospirosis, melioidosis, invasive salmonellosis and \nmalaria, at high sensitivity and specificity, both analytically and clinically.
Author: Ali, Mohammad Ridhuan Mohd
Institution: IMU University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6999543596