Background: According to the important of SALL4 gene during the development of embryonic neurvous system, our aim in this study was to analyze and quantify mRNA expression of SALL4 in Prosencephalon during different stages of chicken embryogenesis.

Materials and Methods: In this experimental study, incubated Ross fertilized eggs were applied in 37°C-37.5°C in 60-65% humidified atmosphere after beginning of embryogenesis. Prosencephalon part of the brain tissue was collected from the eggs, daily. Total RNA extraction and cDNA synthesis was performed from resected tissues. The synthesized cDNA was used as template for quantitatively analysis of SALL4 mRNA expression by real-time PCR.

Results: The Results indicate that the level of SALL4 gene expression is significantly variable during embryogenesis. However it doesn’t show variation during the early days. The maximum copy number of SALL4 mRNA was quantified on 15 th day of chicken development.

Conclusion: SALL4 mRNA expression is high when the Prosencephalon is under development, using of HAMBURGER–HAMILTON chart, there is relation between increasing SALL4 expression and developing limbs and anterior brain.

Background: Brucellosis is a major cause of zoonosis disease and is endemic in hamadan Province in Iran. The purpose of this study was to isolate Brucella species from brucellosis patients and identify different species of this bacterium in order to determine the prevalence of the species.

Materials and Methods: This study was descriptive- cross sectional and fifty blood samples were obtained from brucellosis patients with clinical symptoms. The samples were cultured in BACTEC system and incubated for 14 days. Then, the samples were cultured on Brucella agar and biochemical tests were done for identification of bacteria. Finally, Polymerase Chain Reaction (PCR) applied for confirmation and isolated identification with specific primers.

Results: Seven Brucella strains were isolated from 50 blood samples of the patients with brucellosis by blood culture and PCR. The PCR results on blood specimens showed 4 positive in spite of the negative results of blood culture. PCR and biochemical methods revealed that all the 11 isolated bacteria were Brucella Melitensis.

Conclusion: This study was designed to evaluate PCR technique as a diagnostic tool for brucella spp in comparison to conventional techniques. This study showed a high prevalence of brucellosis due to Brucella Melitensis in Hamadan Province and efforts in this region should be aimed at the eradication of this bacterium.

Background: Pseudomonas aeruginosa is one of the most common nosocomial pathogens with high mortality rates. OprD is the major resistance mechanism to carbapeneme antibiotics. The aim of this study was to determine the expression of the genes encoding these efflux pumps using qRT-PCR.

Materials and Methods: This study examined 100 strains of Pseudomonas aeruginosa isolated from patients admitted to various hospitals in the Hamedan. Conventional phenotypic tests were used for identifying the 100 collected samples, then 31 samples were selected based on type of collected specimen and antibiotic susceptibility test i.e. antibiotic disk diffusion method performed for aminoglycoside, quinolone and carbapenem antibiotics. Furthermore, MIC method was performed for imipenem. Finally, RNA was extracted and converted to cDNA for determining the efflux pump genes expression using qRT-PCR.

Results: Among 8 selected antibiotics, the greatest resistance was for levofloxacin (61.2%, n=19) and the lowest one for imipenem (9.6%, n=3). The results of MIC were to imipenem 12 samples (38.7%) resistant, 13 samples (41.93%) intermediate, and 6 samples (19.35%) sensitive. The OprD gene was present in all strains but different expression has been observed. The strains with over expression of OprD gene showed high sensitivity towards carbapenems family antibiotics especially imipenem.

Conclusion: Identifying of bacterial resistance mechanisms is very complicated and extensive due to different mechanisms involved for similar antibiotics. OprD is main cause of attachment to the carbapenems family antibiotics. The more expression of OprD shows the more antibiotic sensitivity.

Background: In 1970, human papillomavirus (HPV) was introduced as the main etiologic factor of cervical carcinoma. Since there is no possibility of detecting the virus and its subtypes using serological methods and cell culture, the molecular methods such as PCR have particular importance in accurate, early and definite diagnosis of the virus. So, in this research, our goal is to use a proprietary PCR assay based on L1 gene of human papillomavirus for molecular recognition of HPV and to evaluate its prevalence in patient samples.

Materials and Methods: In this experimental study, after collecting of samples from malignant cervical lesions, the viral DNA was extracted from paraffin blocks of 50 clinical samples and PCR was done by specific primers for L1 gene of human papillomavirus in all samples. After the analysis of PCR products by 2% agarose gel electrophoresis, sensitivity and specificity of the test were also evaluated.

Results: Among 50 patient samples, 33 cases were confirmed to be positive for HPV infection and 17 cases were negative, showing high frequency of HPV in this patient population (about 66%). The results of specificity assay were positive for papilloma samples and sensitivity of the assay was 20 copies of recombinant construct containing L1 per reaction.

Conclusion: This study showed that PCR by specific primers for L1 gene of human papilloma virus is a proper and accurate method for detection of this virus and the results confirm the previous reports of correlation between HPV and cervical carcinoma.

Background: Q fever is a zoonotic agent that is endemic in the many parts of the World. It has animal origin as considered as an emerging and re-emerging zoonose in many countries, including Iran. Cattle, sheep, and goats are the primary reservoirs for Q fever. Organisms are excreted in milk of infected animals. This study was conducted to determine the prevalence rate of Coxiella burentii in raw samples obtained from sheep in Bonab.

Materials and Methods: This study was carried out from Autuman 2014 to Winter 2014. Overall, 120 milk samples were collected from 100 dairy cattle breeding complexes and the diagnosis of Coxiella burnetii was confirmed by Nested-PCR method.

 Results: In this study, in total, 26 samples (21.66%) were found to be positive for the presence of Coxiella burnetii.

 Conclusion: Considering the importance of the bacterium, Coxiella burnetii, rapid and accurate diagnosis is of great significance. Due to high accuracy and high speed, molecular techniques are mostly effective in the diagnosis. Thus, the localization of molecular techniques in the diagnosis of Q fever is highly recommended. The results indicated that Cattle's milk could be a potential reservoir of Coxiella burnetii in Iran.

Background: Diabetic retinopathy (DR) is the complication of diabetes mellitus (DM) and causes blindness among adults. Chronic extra cellular hyperglycemia in diabetes stimulates reaction oxygen species ROS production and increases oxidative stress. GPX-1 that was coded by GPX-1 gene is a key enzyme in protecting vessels against oxidative stress. The aim of this study was to evaluate the association of GPX-1 gene Pro 198 Leu polymorphism in patients with diabetic retinopathy.

Materials and Methods: In this case-control study, 160 blood samples of participants including 80 patients with diabetic retinopathy and 80 healthy individual were tested. Genotyping of GPX-1 gene was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) by ApaI enzyme. Data analysis was performed using MedCalc (12.1) program.

Results: The genotype frequencies of the GPX-1 in DR patients for Leu/Leu, Leu/Pro, Pro/Pro were 10%, 62.5% and 27.5%, respectively, while for the control groups were 10%, 70% and 20%, respectively.In ohter words, Ile/Pro heterozygote was the most frequent genotype in patients and controls. According to the results of this study, there was not significant difference between patients with diabetic retinopathy and controls(p=0.52).

Conclusion: It is concluded that GPX-1 gene Pro 198 Leu polymorphism is not associated with DR. Further research is required to clarify the role of GPX-1 gene in DR in Rasht population along higher sample size.

Background: ANKK1 (ankyrin repeat and kinase domain containing 1) gene is a member of the serine/threonine kinase family. This family involved in signal transduction pathways. This gene contains Taq1A (rs1800497) single nucleotide polymorphism. The A1 allele carriers of TaqIA polymorphism have shown reduced DRD2 (Dopamine Receptor D2) receptors. This decrease predisposes individuals to seek for addictive substances to compensate this deficiency in dopaminergic system. The present study investigated TaqIA (rs1800497) polymorphism in heroin and methamphetamine addiction.

Materials and Methods: In this case-control study, 91 male methadone-maintained heroin and methamphetamine addicts and 100 male healthy controls were studied. Genomic DNA extraction was carried out from peripheral blood through salting-out method and individuals were genotyped for TaqIA polymorphism by RFLP-PCR technique and TaqI enzyme was used for RFLP.

Results: This survey revealed the significantly higher frequency of the A1 allele of TaqIA polymorphism in patients than control individuals (p<0.001). The frequency of A1 allele in patient and control individuals was %51 and %22.5, respectively. The A1A1 genotype was detected in 25% of patients and 7% of controls (p<0.001, OR=9.7, 95% CI=3.64-25.85).

Conclusion: The results of this study revealed that the A1 allele of TaqIA polymorphism is significantly associated with heroin and methamphetamine addiction.

Background: Circulating microRNAs are promising biomarkers in diagnosis and assessment of cancerous patients. Quantitative Real-time PCR assay is a sensitive test for evaluating the levels of miRNAs expression. Nevertheless, there is no concurrence on selecting appropriate reference genes for qPCR analysis of miRNAs in circulation. Therefore, the current study aimed to select a suitable reference gene for normalizing the RT-qPCR assay results in plasma samples of patients with gastric cancer.

Materials and Methods: Based on previously published studies, three molecules SNORD47, U6 RNA, and miR-103 were selected as the candidate reference genes. After RNA extraction from plasma samples of 40 patients with gastric cancer and 40 healthy individuals, expression levels of these molecules were evaluated using Real-time PCR method.

Results: The results showed that the developed assays are able to diagnose their specified targets by a suitable linear range. By comparing patients and control groups, although the expression levels of miR-103 molecule were not equal between the two groups (p= 0.017), SNORD47 and U6 RNAs had similar expression levels. However, the variations of SNORD47 expression were lower that U6 RNA.

Conclusion: Based on the results of the current study, the SNORD47 molecule has a stable expression levels in plasma samples of patients with gastric cancer and normal individuals and can be used as an appropriate reference gene for normalizing the quantitative data of qPCR assay.

Background: Niemann-Pick disease (NPD) refers to a group of lysosomal storage diseases that causes abnormal metabolism of lipids. One of the genes that play a role in the pathogenesis of this disease is SMPD1. To date, more than hundred disease- causing mutations have been identified in SMPD1 gene. Due to the large number of mutations in this gene, direct analysis of the mutations is costly and time-consuming. Therefore, indirect linkage analysis using polymorphic markers as an alternative method for molecular diagnosis of the mutations has been recommended. In the present study, allele frequency of rs1542705 genetic marker was analyzed in the Iranian population. The aim was to determine the polymorphic information content (PIC) and the possibility of its application in indirect diagnosing of NPD.

Materials and Methods: After bioinformatics analysis of the SMPD1 gene region, rs1542705 marker was selected for genotyping in Isfahan population. In order to calculate the allele and genotype frequency of the marker, molecular tests were done on 113 DNA samples of unrelated healthy individuals by using ARMS-PCR technique. Finally, the information related to the genotype of the individuals was statistically analyzed using Powermarker and Genepop software.

Results: The analyses showed that the studied population was in accordance with Hardy-Weinberg equilibrium. Allele frequency of rs1542705 marker for T and C alleles was 71.24% and 28.76%, respectively, and the heterozygosity of the marker was 43.36%. Also, polymorphic information content (PIC) was 0.325.

Conclusion: The results of this study showed that rs1542705 marker could be considered as an informative marker for molecular diagnosis of Niemann- Pick disease using linkage analysis in the studied population.

Abstract

Background:  Hydatidiform Mole is a benign trophoblastic tumor is made of ectopic pregnancy. Abnormalities in the number or structure of chromosomes are causes of Hydatidiform Mole common numerical disorders resulted from proliferating repetitive sequences markers as called STR were studied in the region of chromosome X, Y, 13, 18 and 21. This study aimed to investigate chromosomal disorders prevalent in women with hydatidiform mole that was performed using QF-PCR techniques.

Materials and Methods: In this study, 50 women with hydatidiform mole and 80 healthy women as controls were selected. For studying the chromosomal abnormalities resulted of proliferating STR, Chromo Quant QF-PCR kit was used. Polymerase chain reaction was performed in PCR machine. Then electrophoresis was performed on Genetic Analyzer. Finally, amplified fragment were analyzed by Gene Marker software Statistical analysis was performed using SPSS version 19, and t-test. Data were expressed as mean ± SD. In this test, p <0.05 represents significant level between two groups.

Results:  In this study ،of 50 samples, 8 samples of 47XXY (16%), 40 samples of trisomy 21 (80%) and 2 cases of trisomy 18 (4%) were identified.

Conclusion: Anomalies Trisomy 21 (41 ± 1.58) and 47XXY (9.62 ± 1.36) are significantly associated with mydatidiform mole disease (p <0.001). The highest percentage of samples with trisomy 21 and 47XXY had the disease. So, it indicates that these anomalies have the highest percentage in the disease.

Abstract

Background: Candida albicans is still the main etiologic agent of candidiasis. However, infections of non-albicans Candida species are increasing. Candida dubliniensis is similar to C. albicans phenotypically and must be identified due to the better management of infection. The aim of the present study is to defferentiate and identify Candida species by Duplex PCR for getting an epidemiological data of Candida species among clinical specimens.

Materials and Methods: DNA was extracted using phenol-chloroform method from fresh colonies. Internal Transcribed Spacer region was amplified by polymerase chain reaction using specific primers. Based on differences of bands sizes on agarose gel electrophoresis, species were identified.

Results: Ninety four out of 100 patients (49 males and 51 females) had predisposing factors in the present study. Diabetes (73.4%), use of antibiotic (6.3%), vitamin deficiency (4.3%) were the main predisposing factors. The most specimens belonged to mouth (75%), vagina (5%), and blood (4%). All isolates were identified as C. albicans.

Conclusion: Duplex PCR is a rapid and precise method for the detection and differentiation of Candida species carefully, and in this method, phenotypic tests like germ-tube and chlamydoconidia production, as well as biochemical tests are not required for clinical laboratories that have limited resources and time for response to the patients, and it can replace with the traditional methods.

Abstract

Background: Ureaplasma and M. genitalium species belong to a kind of bacteria that are sexually transmitted and are the possible cause of pelvic inflammatory disease and nongonococcal urethritis, and et al. The aim of this study was to determine the urea plasma and Mycoplasma genitalium species frequency in women with vaginal infection and various sexual partners who referred to women^, s health promotion and treatment center in Arak.

Materials and Methods: Endocervical swab samples from 110 women with vaginal infections referred to women^’s health promotion and treatment center in Arak, were prepared. Patients’ personal information and identities during reception process were registered. The samples were transferred to the laboratory in the transport environment and after DNA extraction, were evaluated according to Real-time PCR assay.

Results: Urea plasma and Mycoplasma genitalium bacteria existed in 96(87.27%) and 4(3.63%) of patients, respectively. Among them, 4 cases had both bacteria infections. The amount of isolation in young women between 30-39 years old was more than others.

Conclusion: The results show that the colonization of urea plasma species in adult women is 40-80% and in studied group is 87.27%. These results indicate that with due attention to the increasing number of sexual partners and the increase of sexual activity, the urea plasma colonization of women will increase. In view of the potential influence of mycoplasma species on side effects resulted from pregnancy infection of mothers and mortality, on-time diagnosis and treatment will be increasingly essential.

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.

Abstract

Background: Multiple sclerosis (MS) is a chronic inflammatory disorder described by central nervous system (CNS) demyelination and axonal damage. While the cause of MS is still unknown, it is extensively accepted that novel drug targets need to attention. Retromers are protein complex that have an essential role in endosomal trafficking, and retromer dysfunction has been associated to several neurological disorders. Therefore, this study aimed to compare the expression of SNX2 gene as a part of retromer complex in MS patients with health individuals.

Materials and Methods: In this case-control study, 50 samples of cases of multiple sclerosis (MS) and 50 healthy controls were enrolled. Followed verifying disease, 3cc peripheral blood was given from all subjects. Total RNA was extracted and complementary DNA (cDNA) was synthesized. The relative gene expression was determined using quantitative real-time RT PCR (qRT-PCR) and evaluated by  method.

Results: The expression of SNX2 gene was lower in MS patients compared with healthy controls and it was statistically significant (p< 0.05).

Conclusion: Our study showed that the expression of SNX2 is lower in multiple sclerosis disorder. Considering the functional role of SNX2 as a protein involved in trafficking process, SNX2 may affect receptor function or drug targeting. Therefore, supplementary studies should be done to elucidate the exact mechanism of action of the gene in cellular trafficking.