Background: Bovine Leukaemia Virus (BLV) is the cause of Enzootic Bovine Leukosis (EBL) which belongs to retroviruses including Human T-cell leukaemia virus and simian T-lymphotropic virus. Due to this familiarity, the possibility of BLV transfer from animal production to humans may exist. Materials and Methods: In the present study, formalin-fixed, paraffin-embedded and fresh human lymphomas tissues were used for detecting the BLV Tax genome and BLV Tax expression using nested-PCR and RT-Nested-PCR. Results: Nested-PCR evaluation showed that 9 of 41 samples contained Tax region of BLV genome, of which eight samples belonged to high grade diffuse large cell lymphoma (B-cell type). Also, the results of RT-Nested-PCR showed the BLV Tax expression in 2 of 5 high grade diffuse large cell lymphoma (B-cell type) samples. Conclusion: These findings explain the possible relation between BLV infection and the occurrence of some types of human lymphomas for the first time.

Molecular detection techniques are believed to be key tools for both prevention and treatment follow up of brucellosis within live stock and human beings. Consequently rapid, reliable, easy to perform and automated systems for Brucella detection are urgently needed to allow early diagnosis and adequate antibiotic therapy in time. Brucellosis is a worldwide re-emerging zoonosis causing high economic losses and severe human disease. In attempt to improve current molecular detection of Brucella melitensis, we compared a conventional PCR with PCR- ELISA, to detect brucella genome within standard strains and clinical isolates. Primer sets based on “omp-31” sequence of B. melitensis 16M were designed. The primer specificity was checked with appropriate online bioinformatics softwares. The primer specificity was also confirmed by testing the reaction with non-Brucella strains. A biotinylated probe complementary to an internal sequence of the PCR products was designed. The labeled non-specific fragments bound to streptavidin-coated wells, saturating the solid phase streptavidin by biotin-streptavidin interaction. Compared with conventional PCR, the PCR- ELISA proved to have more sensitivity for Brucella genome after appropriate optimization. Few human serum, whole blood and also different affected tissue samples from slaughtered livestock with brucellosis were used for protocol evaluation. Further samples should be tested before final conclusion about the results.

Introduction: Prcimplantalion genetic diagnosis (PGD) was introduced in the nineties of last century to prevent terminations of pregnancy for fetuses affected by monogenic diseases. Embryos obtained in vitro are biopsied, and the biopsied blastomeres are used to establish a genetic diagnosis, after which only the embryos free of the disease under consideration are transferred into the womans womb. In this presentation, a brief history of technical evolutions in PGU for monogenic diseases will be outlined, followed by an overview of our own and international results. Materials and methods: All tests used to dale are PCR-derivcd: the earlier PGDs involved two rounds of PCR followed by analysis on ethidiumbromide-stained gels. this type of tests shows a high rate of inaccuracy mainly due to ADO, which led to two documented misdiagnoses. Because of this, it was replaced at the end of the nineties by fluorescent PCR followed by analysis on automated sequencers. In fluorescent PCR, the primers used to initiate the PCR reaction are labelled with a fluorochrome, so that the PCR products obtained are also fluorescently labelled, "these PCR products can then he used for fragment analysis on an automated DNA sequencer. Fragment analysis can be carried out directly for e.g. short deletions or insertions, or for point mutations after cutting with restriction en/ymcs. Because of the high efficiency of fluorescent PCR as compared to non-fluorescent PCR, the ADO rate is significantly lower, and thus the misdiagnosis risks were substantially decreased. Fragment analysis is also useful for PCiD using linked microsatellite markers. The combination of mutation analysis combined with linked markers was the next important development that led to a more accurate diagnosis, because now ADO could be determined. More recently, other types of DNA analysis already in use in routine DNA diagnosis, such as minisequencing and sequencing were introduced in PCiD. Finally, (lie latest development in single cell analysis is (lie use of whole genome amplification. Because the total DNA from one blastomere can be amplified to DNA at the micrograin level, this pre-amplified DNA can then be used for more than one application, e.g. for karyotyping with comparative genomic hybridisation (CCil-1) and a monogenic disease with mutation and several markers simultaneously. Results and discussion: At our centre, we have a large experience with different types of PCiD for monogenic diseases, and illustrative examples will be given during the presentation, (lie overall experience in PGD for monogenic diseases of the Centres for Medical Genetics and Reproductive Medicine of the A7-VLJB will be shown. International data as obtained through the LSHRR PGD Consortium will also be discussed. In conclusion, PGD is constantly evolving as far as (lie genetic analysis part is concerned, and closely follows the new technologies used in oilier brandies of DNA analysis. It is to be hoped that further technical improvements will allow practitioners to obtain all necessary results from just one biopsied blastomere. thus insuring that the viability of the embryo is secured as much as possible.

Background: Leishmaniasis is a protozoan parasitic disease and a global health problem. The aim of this study is to diagnose the parasitic infection in humans for epidemiological identification and providing control programs using proprietary co-designed primers in three species of Leishmania.Materials and Methods: 30 common Leishmania isolates were gathered from different centers in Iran. Having been cultured in RPMI-1640 Medium, DNA was extracted and the gene ITS2-rRNA was amplified by PCR. The amplicons were examined by electrophoresis on agarose gel 2%. Also, in FLASH PCR method, a specific probe and florence colour were used to investigae the amplicon existence on sample.Results: The results of the investigations by PCR and FLASH PCR methods show that these methods are sensitive and specific for diagnosis of Leishmania.Conclusion: In this study, identification of Leishmania parasite using specific primer pairs was successful and TaqMan could be one of the most sensitive diagnostic methods to identify parasite load for the ITS2 region of Leishmania.

Introduction: One of the goals of modern genetics is to develope safe prenatal diagnostic tests which do not constitute any risk to the fetus. One potentially non-invasive approach for doing so is to obtain fetal material from the maternal circulation. In this study we obtained Rlaternal blood from the pregnant women and tried to determine sex of fetus by nested-PCR. Material and Methods: To determine the sensitivity of the PCR methods, artificial samples were prepared first by mixing whole blood of adult males with that of the adult females. After performing DNA extraction, PCR was optimized on these samples. Whole blood samples were then obtained from 70 pregnant women in 8 to 12 weeks of gestation after considering the medical ethics. In this study we tried to extract the DNA of the white blood cells (WBC) as well as free DNA of serum and plasma of the maternal bloods and performed Nested-PCR using primers flanking the specific-sequence of V-chromosome or Sex determining region on Y (SRY). If the fetus was male, the size of amplified fragment of the first round product was about 470 base pairs and second round was 254 base pairs. Results: After delivery, Only 32 out of 70 of the samples could be followed and results showed that 18 were male. However, the results of the nested-PCR examination of the serums indicated that 21 of the fetas were male and examination of the WBCs showed that 22 were male, in each group 3 and 4 cases were misinterpreted respectively. So the accuracy of sex determination of the samples in serums and WBCs were 90.62 % and 81.25% respectively which is almost similar to the recent reports (91.5%). We could not obtain good results from the free DNA of the plasma. Conclusion: We have obtained relatively positive results from the analysis of maternal blood and if we could follow all cases to see the results of all deliveries, we could then reconfirm the results of our research. Anyway we hope this project could be able to introduce this new approach as means of noninvasive prenatal diagnosis for detection of common inherited diseases in Iran.

Background: The clinical importance of yeast infections has increased in recent decades. There are 10-15 pathogenic Candida species. The current morphological and physiological methods for identification of Candida species are generally not easy to interpret and may be expensive or time-consuming. In the present study, we introduce and use a new approach for the identification and differentiation of medically important yeast species of Candida. In this method, size polymorphism of the internal transcribed spacer regions, ITS1 and ITS2, of the ribosomal DNA in various Candida species is used as the basis of species recognition.Methods: The genomic DNA of 31 standard strains and 60 clinical isolates was extracted and PCR-amplified using two primer pairs (ITS1-ITS2 and ITS3-ITS4) separately. Both PCR products were mixed and analyzed after standard agarose gel electrophoresis. The species of the tested yeasts were identified by the electrophoretic patterns of the mixed PCR products of each sample, comparing the data obtained from the sequence analyses of ITS1 and ITS2 molecules.Results: By this method, with the exception of C. albicans and C. dubliniensis, we were able to clearly differentiate nearly all common pathogenic Candida species, including C. albicans, C. glabrata, C. gulliermondii, C. parapsilosis, C. tropicalis, C. krusei, C. kefyr, C. lusinaniae and C. rugosa. All standard and clinical strains were identified correctly, without expensive methods such as sequencing and capillary electrophoresis. Conclusion: It seems that the PCR-FSP method introduced in this study is the easiest molecular approach for the identification of a wide range of pathogenic Candida species and is applicable for diagnostic and epidemiological purposes in reference laboratories.

To diagnose brucellosis in cattle, DNA was extracted from 50 bovine blood samples with positive serologic test results and from 45 bovine blood samples with negative serologic test results. This DNA was used for amplification of 31 KDa antigen gene by polymerase chain reaction (PCR). PCR product was analyzed by 2% agarose gel electrophoresis. Thirty out of 50 (60%) bovine blood samples with a positive serologic test showed positive PCR results and 20 out of 50 (40%) had negative PCR results. Meanwhile, 12 out of 45 (26%) bovine blood samples with negative serologic test showed positive PCR results. Results showed that the PCR was not able to detect all positive brucellosis. Therefore, combination of PCR and serologic tests is suggested. Both PCR and serologic tests had false results. Milk and vaginal secretion samples for doing PCR could be suggested.