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Optimization of a loop-mediated isothermal amplification method for a detection of Haemophilus influenzae infection.
Zadáková, Kateřina ; Jeřábek, Petr (advisor) ; Svášková, Dagmar (referee)
Haemophilus influenzae is a gramnegative, rod-shaped and predominantly human pathogen. Infection caused by this microorganism is especially dangerous for children under five years of age, who are mainly at risk of pneumonia and meningitis.Althought vaccination is available against its most dangerous type b, it still causes problems, especially in developing countries. Due to the insufficient vaccination, strains that are more resistant to the antibiotics are more spreading in these countries. Equipment requirement of current detection options for H. influenzae infection(e.g. polymerase chain reaction) causes, that reliable detectionis a problem in poor countries. Loop-mediated isothermal amplification could be a cheap and reliable method. The aim of this bachelor thesis was the design of sets of primers and their applicationinthe LAMP reaction. The designed primers targetedthe ompP6 gene, which was alreadythe target of LAMP reaction in some studies and is also a frequent target in PCR detection.As a part of the optimization, different volumes of the reaction mixtures and multiple detection options were tested -colorimetric detectionusinganacid-base indicator and fluorescence detection using different dyes monitored in real time. Key words: Haemophilus influenzae, loop-mediated isothermal...
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