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Okužbe s humanim virusom citomegalije (CMV) so nevarne predvsem za imunsko oslabele gostitelje, kot so prejemniki transplantatov, bolniki s sindromom pridobljene imunske pomanjkljivosti oz. gostitelji z nedozorelim imunskim sistemom. Za zdravljenje in preprečevanje okužb pri ogroženih skupinah se najpogosteje uporablja protivirusno zdravilo ganciklovir. Resne zaplete povzroča pojav odpornosti proti zdravilom, ki so posledica mutacij na genih UL97 in/ali UL54. Natančno zaznavanje in kvantifikacija mutacij je ključna za hitro ukrepanje in prilagajanje zdravljenja. Ker digitalna verižna reakcija s polimerazo (dPCR) omogoča zelo natančno in točno kvantifikacijo redkih polimorfizmov posameznih nukleotidov, njena uporaba na tem področju narašča. V okviru magistrske naloge smo razvili metodo za zaznavanje mutacije L595S (metoda L595S), ki nastane na genu UL97 in povzroča odpornost proti zdravilu ganciklovir. Za razvoj metode smo uporabljali sintetično DNA, ki je bila hibrid dveh zaporedij virusa CMV, dela zaporedja UL97 in dela zaporedja UL54. Metodo L595S smo razvijali in ovrednotili s PCR v realnem času (qPCR) in dPCR, rezultate pa primerjali z delovanjem že ovrednotene metode, ki omogoča natančno in zanesljivo zaznavanje CMV. Dokazali smo, da metoda L595S omogoča zaznavanje majhnih deležev mutacije v ozadju divjega tipa ter da je dPCR bolj občutljiva za zaznavanje mutacije v primerjavi s qPCR. Human cytomegalovirus (CMV) is a pathogen that can be life-threatening in immunocompromised patients, such as transplant recipients, patients with AIDS and in patients with immature immune system. Virostatic ganciclovir is currently the principle drug used for treatment or prevention of CMV disease. Development of antiviral resistance is a serious complication of CMV virostatic therapy caused by mutations in UL97 and/or UL54 genes. Accurate quantification and rapid detection of gene mutations is crucial for optimization of treatment decision making. Due to its precision and accuracy, digital polymerase chain reaction (dPCR) is being increasingly used for detection and quantification of mutations, including single nucleotide polymporphisms. Within the framework of this master thesis we developed an assay for the detection of L595S mutation (L595S assay), which may emerge in UL97 gene and causes ganciclovir resistance. For the development of the L595S assay a sintetic DNA, hybrid of UL97 and UL54 sequences, has been used. The assay has been developed and characterised on both dPCR and real-time PCR (qPCR) platforms, and the results were compared to a well-characterised assay for accurate detection of CMV. We concluded that the here developed assay can distinguish L595S mutation in a very high background of wild-type sequences and that dPCR is a more sensitive method compared to qPCR for this purpose. |
