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Title:
PRIMERJAVA RAZLIČNIH MOLEKULARNIH METOD ZA DIAGNOSTIKO CELIAKIJE
Authors:
ID
Hrovat, Neja
(Author)
ID
Potočnik, Uroš
(Mentor)
More about this mentor...
Files:
UNI_Hrovat_Neja_2013.pdf
(8,74 MB)
MD5: 2D0DF8A31DED1D42AFD0A28A96A1080F
Language:
Slovenian
Work type:
Bachelor thesis/paper
Typology:
2.11 - Undergraduate Thesis
Organization:
FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:
Celiakija je ena izmed pogostejših imunskih bolezni, ki se razvije pri vnosu glutena pri genetsko dovzetnih posameznikih; poleg okoljskih dejavnikov k razvoju celiakije prispeva tudi genetska predispozicija. Večina bolnikov nosi genotip človeškega levkocitnega antigena HLA-DQ2 ali HLA-DQ8. To so heterodimerni proteini, katere najdemo na površju antigen predstavljajočih celic. Za diagnozo celiakije je potrebnih več preiskav. Med njimi so biopsija tankega črevesa, opazovanje stanja ob prehodu na brezglutensko dieto, serološke preiskave in genetski testi. Evropsko združenje za pediatrično gastroenterologijo, hepatologijo in prehrano (ESPGHAN) je za diagnostiko celiakije priporočilo določanje prisotnosti genetskega zapisa za HLA-DQ2 in HLA-DQ8, saj bolniki s celiakijo nosijo ta genetski zapis. Na podlagi tega smo se odločili za primerjavo dveh metod genetske diagnostike, SSP PCR in rt-PCR. SSP PCR je verižna reakcija s polimerazo z uporabo sekvenčno specifičnih začetnih oligonukleotidov; za to metodo smo uporabljali komercialni kit Olerup. rt-PCR pa je verižna reakcija s polimerazo v realnem času, kjer merimo količino produkta v vsakem ciklu med samo reakcijo, z merjenjem fluorescence. Za to metodo smo uporabljali komercialni kit Eligene. Analiza je bila opravljena na šestih vzorcih, od tega so štirje bolniki s celiakijo, potrjeni v slovenskih splošnih bolnišnicah. Rezultati obeh diagnostičnih testov se ne skladajo. Ugotovili smo, da se rezultati Eligene diagnostičnega kita skladajo s potrjenimi diagnozami postavljenimi v splošnih bolnišnicah, medtem ko za Olerup kit nismo potrdili diagnoz. Primerjava zahtevnosti izvedbe posameznega kita je pokazala, da je diagnostika z uporabo rt-PCR metode časovno manj zahtevna od SSP PCR metode in uporabniku bolj prijazna, saj zahteva manj priprave. Interpretacijo rezultatov uporabnik zlahka opravi sam. Slabost, ki bi jo izpostavili je, da potrebujemo real time aparaturo, ki je cenovno težje dostopna. Izvedba diagnostike z Olerup kitom se je izkazala za časovno bolj zahtevno kot diagnostika z Eligene kitom. Od uporabnika zahteva več časa za pripravo, izvaja se v 2 korakih. To sta reakcija PCR in analiza fragmentov na agarozem gelu. Pozitiven je razvoj programske opreme ki omogoča analizo, vendar je ta toliko bolj zahtevna, da je za interpretacijo rezultatov potrebna strokovna pomoč v podjetju šolanega osebja. V kolikor bi se morali odločiti med tema dvema kitoma, bi se odločili za diagnostiko rt-PCR z Eligene kitom.
Keywords:
celiakija
,
PCR v realnem času
,
sekvenčno specifični PCR
,
genetska diagnostika
Place of publishing:
Maribor
Publisher:
[N. Hrovat]
Year of publishing:
2013
PID:
20.500.12556/DKUM-41288
UDC:
575(043.2)
COBISS.SI-ID:
17477910
NUK URN:
URN:SI:UM:DK:QWOE25YQ
Publication date in DKUM:
18.09.2013
Views:
3792
Downloads:
237
Metadata:
Categories:
KTFMB - FKKT
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:
HROVAT, Neja, 2013,
PRIMERJAVA RAZLIČNIH MOLEKULARNIH METOD ZA DIAGNOSTIKO CELIAKIJE
[online]. Bachelor’s thesis. Maribor : N. Hrovat. [Accessed 29 March 2025]. Retrieved from: https://dk.um.si/IzpisGradiva.php?lang=eng&id=41288
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Secondary language
Language:
English
Title:
COMPARISON OF DIFFERENT MOLECULAR METHODS FOR THE DIAGNOSIS OF CELIAC DISEASE
Abstract:
The celiac disease is one of the most common autoimmune disorders, occurring when gluten is ingested by genetically predisposed individuals; alongside environmental factors, a genetic predisposition contributes to the development of the celiac disease. Most of the individuals, suffering from this illness share either the HLA-DQ2 or HLA-DQ8 human leukocyte antigen genotype – the heterodimeric proteins, found on the surface of antigen-presenting cells. Several medical examinations must be performed before a diagnosis can be made. Among them are: small intestine biopsy, controlled trial of gluten free diet, serological tests, and genetic testing. To determine whether an individual is prone to the disease, The European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) has recommended identifying the HLA-DQ2 and HLA-DQ8 genotypes, present in affected individuals. Based on this information, we have decided to compare two genetic diagnostic methods: The sequence specific primer polymerase chain reaction (SSP PCR), and real-time polymerase chain reaction (rt-PCR). The first is a polymerase chain reaction (PCR) where sequence specific primers – oligonucleotides are used, while the latter is a PCR, where the quantity of a product is measured in between every cycle of the reaction by measuring fluorescence. With the first method, Olerup diagnostics kit was used, while Eligene diagnostics kit was used with the second. The analysis was conducted on six samples, of which four belonged to hospital patients, admitted for celiac disease. The results of both diagnostic tests do not match. We have concluded that the results, provided by the Eligene diagnostic kit, are in accordance with the medical diagnoses, while the same conclusions could not be made for the results, provided by Olerup. A comparison of the difficulty of implementation of individual detection kits has determined that the rt-PCR method is less complex to implement and more user friendly than the SSP PCR method, as it requires less preparation. The results can easily be interpreted by a single individual. Here, the only disadvantage is that a more expensive real-time machine is required. Diagnostics has proven more time consuming, using the Olerup kit; its implementation requires more time to prepare. It is conducted in two stages, the PCR reaction and the agarose gel fragment analysis. Even though the software equipment needed for the analysis is highly developed, the analysis itself is difficult to perform and requires professional assistance. If we were to choose between the two kits and methods, we would have chosen the rt-PCR method, using the Eligene kit.
Keywords:
celiac disease
,
real-time polymerase chain reaction
,
the sequence specific primer polymerase chain reaction
,
genetic diagnostics
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