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ID naloge: 56 Letnik: 1999 Predmet: mikrobiologija in imunologija
Uporabnost verižne reakcije s polimerazo (PCR) v diagnostiki gonoreje
Avtor: Borut Gubina, Jernej Šavs
Mentor: doc. dr. Mario Poljak, dr. med
Somentor: as. mag. Marko Potocnik dr. med., dr. stom. in asist. dr. Katja Seme, dr. med.
Uvod: Neisseria gonorrhoeae je po Gramu negativni diplokok, ki povzroca spolno prenosljivo bolezen gonorejo. Kljub temu, da je bolezen poznana že dalj casa in da je raziskana patofiziologija, diagnostika in zdravljenje te bolezni, ostaja sopotnik ljudi. Prenaša se najveckrat s spolnimi odnosi med nestalnimi partnerji, z okužene matere na otroka in z enega okuženega partnerja na drugega.
V diagnostiki gonoreje je potrebno uvesti bolj zanesljivo metodo dokaza gonokoka kot je klasicni dokaz z osamitvijo bakterije in nadaljnjo identifikacijo, ki temelji predvsem na fermentaciji razlicnih sladkorjev. Ker serološka diagnostika ni zanesljiva, ponuja uporaba tehnike verižne reakcije s polimerazo (PCR) pomembne prednosti. S to tehniko zanesljivo dolocimo znacilne odseke bakterijske DNA v razlicnih kužninah.
Namen: Ugotoviti uporabnost diagnostike okužb z N. gonorrhoeae s komercialno dostopnim PCR testom COBAS AMPLICOR (Roche Diagnostics Systems, Branchburg, N.J., ZDA) in njegovo zanesljivost v primerjavi s standardno diagnosticno metodo.
Metode: Zbrane vzorce smo razdelili v tri skupine:
· skupina A: 113 klinicnih vzorcev odvzetih v Ambulanti za spolno prenosljive bolezni na Dermatološki kliniki v Ljubljani (skupina z visokim tveganjem za okužbo),
· skupina B: vzorci urina 48 študentov medicine (skupina z nizkim tveganjem za okužbo) in
· skupina C: 31 sevov šestih razlicnih bakterijskih vrst iz rodu Neisseria.
V skupini A smo zbirali prvi curek urina (izpirek secnice) in bris secnice ter maternicnega vratu pri ženskah. Vsak bris smo neposredno cepili tudi na ustrezni gojišci in ga pri 36°C v 5% CO2 atmosferi inkubirali 72 ur. V skupini B smo jemali prvi curek urina. V skupini C smo uporabili zmrznjene, že identificirane seve razlicnih vrst najserij.
Rezultati: V skupini A smo ugotovili tri pozitivne bolnike z metodo PCR in s standardno metodo osamitve gonokokov iz kužnine. V skupini B so bili vsi vzorci negativni. V skupini C smo kot pozitivne opredelili samo tiste seve najserij, teh je bilo deset, ki so pripadali vrsti N. gonorrhoeae.
Sklepi: Z našim delom smo dokazali, da je test COBAS AMPLICOR PCR uporaben za diagnostiko gonoreje, saj je vsaj tako zanesljiv kot klasicna diagnostika in dovolj specificen, da razloci med N. gonorrhoeae in drugimi bakterijskimi vrstami v rodu Neisseriaceae.
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[Abstract / English version]
Aplication of polimerase chain reaction (PCR) in diagnostics of gonorrhea
Author: Borut Gubina, Jernej Šavs
Mentor: doc. dr. Mario Poljak, dr. med
Co-mentor: as. mag. Marko Potocnik dr. med., dr. stom. in asist. dr. Katja Seme, dr. med.
Introduction: Neisseria gonorrhoeae is a Gram negative diplococcus the cause of sexually transmitted disease gonorrhea. Although is has been known to be a human pathogen for a long time and we know the pathophysiology, the diagnostics and the therapy of this disease, it remains an accountable sexually transmitted disease. It is transmitted through a promiscuous intercourse, from infected mother to the newborn and from one partner to another.
It is necessary to implement a more reliable method of diagnostics of gonorrhea as is the classical cultivation and identification of the bacteria, that is based on selective fermentation of different sugars. As the serological diagnostics is not sufficiently reliable, the polymerase chain reaction (PCR) technique carries important advantages. With this technique, we can accurately detect bacterial DNA fragments in different specimens.
Goal: To asses the use of the COBAS AMPLICOR PCR (Roche Diagnostics Systems, Branchburg, N.J., USA) as a diagnostic method for Neisseria gonorrhoeae infection and its reliability in comparison with the classical method.
Methods: We divided the collected samples in three groups:
· Group A group of 113 clinical specimen from the STD Outpatient Clinic, Department of Dermatology in Ljubljana (urine samples, urethral and cervical smears), accounted as a population with high risk of infection,
· group B of 48 medical students (urine rinse of urethra), presumed to be a population with low risk of infection, and
· group C of 31 frozen samples of six different strains from the genus Neisseriaceae that have shown the specificity of the COBAS AMPLICOR PCR test.
In the group A we collected the first void of urine (urethral wash), a urethral and cervical smear. We applied all the smears directly on the specific culture media and incubated them at 36°C in 5% CO2 atmosphere for 72 hours. In the group B we collected the first void of urine. In the group C we used different frozen and already identified strains of Neisseriaceae.
Results: In the group A we found three gonorrhea positive patients with the PCR method, as well as with the standard method of cultures and fermentation of sugars. All the results of the testing in the group B were negative. In the group C we correctly identified the 10 N. gonorrhoeae among the different Neisseriaceae strains.
Conclusions: With our work we proved that COBAS AMPLICOR PCR test is useful for diagnostic and screening purposes. It is as reliable as classical diagnostic method and enough specific to discern N. gonorrhoeae from other bacteria of the genus of Neisseriaceae.
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