Development of real-time fluorescence multiplex loop-mediated isothermal amplification assay for simultaneous detection of Streptococcus pneumoniae and Haemophilus influenzae / Nee Yuan Qi

Nee , Yuan Qi (2018) Development of real-time fluorescence multiplex loop-mediated isothermal amplification assay for simultaneous detection of Streptococcus pneumoniae and Haemophilus influenzae / Nee Yuan Qi. Masters thesis, University of Malaya.

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Abstract

Pneumonia is one of the leading causes of death in children worldwide, accounting for 19% of all deaths of children under the age of five years old. Rapid identification of the causative agents is crucial to ensure prompt treatment is given to reduce infection-related morbidity and mortality rate among hospitalized patients. Current conventional laboratory culturing methods are time consuming and lack of sensitivity and specificity. The loop-mediated isothermal amplification (LAMP) method is an alternative amplification technique which amplifies the genes of interest at constant temperature. It is rapid, highly specific and efficient. Hence, the objective of the study was to develop and evaluate the specificity and sensitivity of two monoplex LAMP assays targeting Streptococcus pneumoniae and Haemophilus influenzae. The developed monoplex LAMP assays were further combined and optimized into a real-time multiplex fluorescent LAMP (mf-LAMP) assay for simultaneous differential detection of the two pathogens. Two sets of species-specific primers targeting lytA (autolysin) gene from S. pneumoniae and pal (outer membrane protein P6) gene from H. influenzae were used and their specificity and sensitivity were evaluated against 59 targeted and non-targeted bacterial strains. In monoplex LAMP assay, positive amplification for S. pneumoniae was found between 30 min – 35 min with DNA concentration ranged between 108 ng-1500 ng per reaction while 17 min - 20 min for H. influenzae strains with DNA concentration between 120 ng – 400 ng per reaction. The detection limit for each of the LAMP assay was also found 6.6 ng per reaction for S. pneumoniae and 32.7 ng per reaction for H. influenzae. Thereafter, a real-time mf-LAMP was constructed by combining these two sets of ten primers in a single reaction with an additional fluorescent-labelled primer termed FIP (Forward Inner Primer) to generate a single-step, closed-tube real-time DNA amplification by detection of fluorescent signal. The mf-LAMP assay was further optimized and validated using 20 strains of S. pneumoniae, 20 strains of H. influenzae and 19 non-targeted strains including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and methicillin-sensitive Staphylococcus aureus. This newly developed mf-LAMP assay showed 100% specificity toward both targeted strains with no cross-reaction with non-targeted strains. However, the time for positive amplification were delayed, 40 - 45 min for S. pneumoniae and 33 - 39 min for H. influenzae detection compared to monoplex LAMP assays. The detection limit was 66 pg per reaction for S. pneumoniae and 32.7 pg per reaction for H. influenzae demonstrating 100 and 1000 time higher sensitivity in detection of S. pneumoniae and H. influenzae respectively compared to monoplex LAMP assays. Moreover, the detection limit for mf-LAMP was equivalent to conventional PCR. Although the cost for generating fluorescent labelled primers was higher, the mf-LAMP assay allows real-time detection of two pathogens in a single-step closed-tube assay that requires short preparation time and smaller amount of reagents in comparison to monoplex LAMP assay. In conclusion, the multiplex LAMP assay developed was a simple and rapid method for identification of pathogens associated with childhood pneumonia.

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