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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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Detectiono fphytopathogenic quarantine bakterium \kur{Erwinia amylovora} using LAMP technique
KLOJDA, Martin
Erwinia amylovora is phytopathogenic bacterium which causes serious plant disease Fire Blight on pome-fruit and surrounding 200 Rosaceae species. Today this disease is spreading across Europe, the Near East, the Mediterranean, New Zealand, the United States and many other countries. Fire Blight is a very destructive disease. Among the symptoms are scorched leaves, necrosis, cankers, secretion of ooze from fruit and other affected location and watering from fruits. Insects like bees can transport bacterium cells into other blossoms and trees. Due to this fact Fire Blight can spread very quickly and destroy harvests, damage trees and even destroy orchards. There is no protection in Europe except resistence breeding, quarantine arrangement, or using antagnonistic bacterium. Early recogntion of Fire Blight symptoms is a key step in the prevention of more severe consequences. There are many tools in diagnosing Erwinia amylovora including the observation of symptoms, cultivation methods, serological or molecular biological techniques among others. In this paper I demonstrate some of these detection techniques. As the quick detection of Erwinia amylovora is important, I summarize the molecular detection technique LAMP (loop-mediated isothermal amplification), which is a quick, low-cost and very sensitive technique besides other methods because of the isothermal conditions for amplification. Because of this, LAMP may be a suitable diagnostic tool for the detection of Erwinia amylovora.
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