Author/Editor		Pislak, M; Ocepek, M; Zabavnik-Piano, J; Pogačnik, M
Title		Primerjava štirih metod osamitve DNA Mycobacterium avium subsp. paratuberculosis iz vzorcev tkiva
Translated title		Comparison of four methods for isolation of Mycobacterium avium subsp. paratuberculosis DNA from tissue samples
Type		članek
Source		Slov Vet Res
Vol. and No.		Letnik 40, št. 2
Publication year		2003
Volume		str. 93-9
Language		eng
Abstract		The PCR reaction (polymerase chain reaction) is a rapid and specific method for detection the IS900 (insertion sequence IS900), which is a DNA sequence specific for Mycobacterium avium subsp. paratuberculosis. However, the efficacy of the method is dependent on the qualitative isolation of DNA from tissue samples. In this paper, four methods of isolation of the Mycobacterium avium subsp. paratuberculosis DNA from 30 intestinal and lymph-node samples, which were taken from 11 seropositive small ruminants that originated from paratuberculosis-positive flocks, were compared. The isolation of genomic DNA was performed by: l. using the commercial kit (High Pure PCR Template Preparation Kit, Roche, Mannheim); 2. treatment the samples with HPC (hexadecylpyridinium chloride) and then performing the isolation using the same commercial kit; 3. the method described by Gwozdz; and 4. treatment of the samples with HPC and then performing the isolation with the method described by Gwozdz. Using the first method of isolation we obtained 22 positive samples, with the second method 24 samples were positive, with the third method 13 samples were positive and with the fourth method 16 samples were positive. The results indicate that the second method, treatment of the samples with HPC and subsequent DNA isolation with the commercial kit (Roche), was the most efficient. This method was also the fastest and the easiest to perform.
Summary		Reakcija PCR (polymerase chain reaction), s katero lahko dokazujemo navzočnost IS900 (insercijske sekvence IS900), to je za Mycobacterium avium subsp. paratuberculosis značilnega odseka DNA, je hitra in specifična, vendar pa je uspešnost le-te na vzorcih tkiv odvisna od kvalitetne osamitve DNA. V raziskavi smo primerjali štiri načine osamitve DNA Mycobacterium avium subsp. paratuberculosis iz 30 vzorcev tkiva črevesja in bezgavk 11 serološko pozitivnih ovc in koz iz črede, okužene s paratuberkulozo. Osamitev genomske DNA Mycobacterium avium subsp. paratuberculosis smo izvedli: 1. z uporabo kompleta za osamitev DNA (High Pure PCR Template Preparation Kit, Roche, Mannheim), 2. z uporabo istega kompleta (Roche) s poprejšnjo obdelavo vzorcev s HPC (heksadecilpiridijevim kloridom), 3. s postopkom, povzetem po Gwozdzu in sodelavcih in 4. s postopkom, povzetem po Gwozdzu in sod., s poprejšnjo obdelavo vzorcev s HPC. Pri prvem načinu osamitve DNA je bilo pozitivnih 22 vzorcev, pri drugem 24, pri tretjem 13 in pri četrtem načinu 16 vzorcev. Iz rezultatov je razvidno, da je bila najučinkovitejša osamitev DNA z uporabo kompleta (Roche) s poprejšnjo obdelavo vzorcev s HPC. Ta način osamitve je bil tudi najhitrejši, izvedba pa najpreprostejša.
Descriptors		PARATUBERCULOSIS MYCOBACTERIUM AVIUM POLYMERASE CHAIN REACTION SHEEP GOATS