magistrsko delo
Anja Papež (Author), Eva Ružić-Sabljić (Reviewer), Darja Keše (Mentor)

Abstract

Mycoplasma pneumoniae povzroča okužbe dihal pri ljudeh vseh starostnih skupin povsod po svetu. Za zdravljenje mikoplazemskih okužb se najpogosteje uporablja makrolidne antibiotike. V zadnjih letih pa je število okužb z odpornimi sevi M. pneumoniae proti makrolidom močno naraslo. Prva poročila o razširjenosti odpornih sevov so se pojavila na Japonskem leta 2000 in se kasneje hitro razširila po celotni Aziji, s časom tudi v Evropo in Ameriko. Rezistenca je povezana s točkovnimi mutacijami v domeni V gena 23S rRNA na mestih 2063, 2064, 2067 in 2617. Najpogostejša mutacija je A2058G (E. coli številčenje), ki ustreza mestu 2063 v genomu bakterije M. pneumoniae in povzroči visoko stopnjo odpornosti. V raziskavi smo želeli pripraviti metodo PCR v realnem času z analizo temperature taljenja za sočasen dokaz okužbe bolnika z bakterijo Mycoplasma pneumoniae in njeno občutljivost za makrolide. Testirali smo 137 vzorcev izolirane DNA bolnikov, pri katerih je bila dokazana okužba z M. pneumoniae z metodo PCR v realnem času in 7 vzorcev DNA M. pneumoniae z že znano mutacijo v genu 23S rRNA, ki smo jo dokazali z metodo pirosekvenciranja. Analiza temperature taljenja je jasno ločila divji sev od mutante. Rezultati testa PCR so bili skladni z rezultati pirosekvenciranja. S testom nismo dokazali nobenega novega seva z odpornostjo proti makrolidom. Občutljivost metode je 10 kopij DNA M. pneumoniae/µL. Dokazali smo 100 % specifičnost testa.

Keywords

mikoplazme;Mycoplasma pneumoniae;makrolidni antibiotiki;odpornost proti makrolidom;PCR v realnem času;analiza temperature taljenja;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL MF - Faculty of Medicine
Publisher: [A. Papež]
UDC: 579.61:579.887:577.182.62:577.2.083
COBISS: 4963960 Link will open in a new window
Views: 1004
Downloads: 375
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Other data

Secondary language: English
Secondary title: Determining the sensitivity of Mycoplasma pneumoniae for macrolides by real-time PCR
Secondary abstract: Mycoplasma pneumoniae causes respiratory tract infection in people of all ages and around the world. Mycoplasma infections are most often treated with macrolide antibiotics. In the last few years the number of infections with resistant M. pneumoniae strains to macrolides has increased. The first reports on the extent of the resistant strains appeared in Japan in 2000, then spread across Asia and after some time surfaced in Europe as well as in America. The resistance is associated with point mutations in the domain of V gene 23S rRNA on locations 2063, 2064, 2067 and 2617. The most frequent mutation is A2058G (E. coli numbering) which fits location 2063 in the bacterial genome of the M. pneumoniae and causes a high level of resistance. In the research we wanted to prepare real-time PCR with melting curve analysis to simultaneously proof the patient’s infection with the bacteria Mycoplasma pneumoniae and its sensitivity to macrolides. Using the real-time PCR we have tested 137 samples of isolated patients’ DNA with proven infection with M. pneumonia and 7 samples of M. pneumoniae DNA with the mutation in gene 23S rRNA, which was proven by the pyrosequencing method. Melting curve analysis clearly separated wild strain from mutation. The PCR test results were in accordance with the pyrosequencing method. However, a new strain with resistance to macrolides was not proven. Method sensitivity is 10 copies DNA M. pneumoniae/µL. We have proven 100 % test specificity.
Secondary keywords: mycoplasmas;Mycoplasma pneumoniae;macrolide antibiotics;macrolides resistance;target gene mutation;real-time PCR;melting curve analysis;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij mikrobiologije
Pages: X, 65 f.
ID: 10977451