Author/Editor     Srovin, Tina
Title     Razvoj nove diagnostične metode za hitro in zanesljivo ugotavljanje mutacije CCR5delta32
Type     monografija
Place     Ljubljana
Publisher     Medicinska fakulteta
Publication year     2001
Volume     str. 47
Language     slo
Abstract     BACKGROUND. Chemokine receptor CCRS, which acts along with CD4 molecule as a major corector for HIV-1 (human immunodeficiency virus) entry into the human cells, plays an important role in the pathogenesis of HIV. In 1996, a 32 bp deletion in the coding sequence of the CCRS (CCR5delta32) was established as the only known natural protection mechanism against sexual and vertical transmission of HIV-1 infection. The detection of the CCR5delta32 is based on time-consuming convetional PCR (polymerase chain reaction) method. Recently developed LightCycler system (Roche Molecular Biochemicals, Germany) represents a new method for rapid and reliable amplification of nucleic acids. LightCycler system is used mainly for the detection of different microorganisms. To the best of our knowledge, only five studies were published until now in which LightCycler system was used for the detection of deletions. Maximal length of deletions detected using LightCyclei system has been 9 bp. AIM. The aim of our study was to develop an entirely new method for rapid and reliable detection of CCR5delta32 using LightCycler system. We also wanted to determine if deletions larger than 9 bp can be detected using this system. In the second part of the study we evaluated the reliability of the new method on 253 DNA samples with known CCRS genotype. HYPOTHESIS. CCR5delta32 can be reliably detected using LightCycler system in 30 minutes. LightCycler is capable to detect deletions larger than 9 bp. METHODS. In the first part of our study we developed a new method for the detection of CCR5delta32. After selection of the most appropriate primers we optimized all amplification conditions. The optimization was performed on three DNA samples with known CCRS genotype. (Abstract truncated at 2000 characters).
Descriptors     HIV INFECTIONS