Association of the ACE rs4646994 and rs4341 polymorphisms with the progression of carotid atherosclerosis in slovenian patients with type 2 diabetes mellitus.

The current study was designed to reveal possible associations between the angiotensin-converting-enzyme (ACE) gene polymorphisms (rs4646994 and rs4341) with markers of carotid atherosclerosis in patients with type 2 diabetes mellitus (T2DM) in a 4-year-long follow-up study. Five hundred and ninety-five T2DM subjects and 200 control subjects were enrolled. Genotyping of ACE polymorphisms was performed using KASPar assays, and ultrasound examinations were performed twice (at the enrollment and at follow-up). With regard to the progression of atherosclerosis in subjects with T2DM, statistically significant differences were demonstrated in the change of the sum of carotid plaques thickness for the rs4646994 polymorphism. We did not demonstrate an association between the tested polymorphisms (rs4646994 and rs4341) and either carotid intima media thickness (CIMT) or CIMT progression in a 3.8-year period. In our study, we demonstrated that subjects with T2DM with the DD genotype of the rs4646994 [ACE insertion/deletion (I/D)] polymorphism had faster progression of atherosclerosis in comparison to subjects with other genotypes.

Comparison of different molecular methods for assessment of equine arteritis virus (EAV) infection: a novel one-step MGB real-time RT-PCR assay, PCR-ELISA and classical RT-PCR for detection of highly diverse sequences of Slovenian EAV variants.

In the present study, a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy with minor-groove-binder (MGB) technology for the detection of EAV from 40 semen samples of Slovenian carrier stallions was tested. A novel MGB probe (EAVMGBpr) and a reverse primer (EAV-R) based on the multiple sequence alignment of 49 different EAV strain sequences of the highly conserved ORF7 (nucleocapsid gene) were designed. The performance of the assay was compared with different molecular detection methods. Three different primer pairs targeting the ORF1b and ORF7 were used, respectively. The real-time RT-PCR assay was at least 2 log(10) more sensitive than the classical RT-PCR and at least 1 log(10) more sensitive than the primer set used in the semi-nested PCR. The specificities of the amplification reactions were confirmed with biotinylated probes in the PCR-enzyme-linked immunosorbent assay (PCR-ELISA). Under the conditions described in our study, the sensitivity of the real-time RT-PCR was found to be superior to the PCR-ELISA assay. Thus, while the PCR-ELISA method was found to be both relatively demanding and time consuming, better sensitivity coupled with high specificity and speed of the assay makes the real-time RT-PCR a valuable tool for diagnosis of EAV infection.