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Background and Objectives:HBV and HTLV-I are life threatening infectious agents in patients who receive blood and blood products. Although serological methods have been proved to be useful, detection of these viruses has remained a challengingissue due to the many obstacles. By the advent of Nucleic Acid Testing methods, especially in multiplex format, more precise detection is possible.The objective of this study was to develop a reliable, rapid and cost- effective method tosimultaneously detect HBV and HTLV-I. Materials and Methods: We have developed a multiplex Real time-PCR assay for simultaneous detection of HBV and HTLV-I. Primer sets were designed for highly conserved regions of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex Real-time PCRs were performed. Results: Analytical sensitivity was considered to be 1000 and 100 copies/ml for HBV and HTLV-I, respectively. High concentration of one virus had no adverse effect on detection of t low concentrations of the other one. By analyzing 30 samples, clinical sensitivity of the assay was determined to be 87% and 96% for HBV and HTLV-I, respectively. Using different viral and human genome samples, the specificity of the assay was verified to be 100%. Conclusions:We have developed a reliable, rapid and cost effective method tosimultaneously detect HBV and HTLV-I.Our results indicatedthe high capability of this simple and rapid method for detecting these viruses in clinical samples.

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Aim: Group A Streptococcus (GAS) is the causative agent of several invasive and non-invasive diseases. Several virulence factors contribute to the pathogenesis of GAS, such as M protein, hemolysins, and extracellular enzymes. Due to the improper use of antibiotics, the resistance of these microorganisms to antibiotics is increasing. Bacteriocins as an alternative to antibiotics are of great importance. In this study, the effect of antimicrobial Bacteriocin nisin was investigated on the expression of smeZ gene.
Materials & Methods: Samples were taken from the site of infection on the skin surface of the patients at the dermatology clinics of Tehran public hospitals. The specimen was immediately transferred to the primary culture medium or basal medium. Chromosomal DNA extraction was performed using the standard method for the extraction of Streptococcus pyogenes genomes. Multiplex PCR was performed to identify the presence of smeZ, speI, and speH genes in the isolates. The expression of smeZ gene was evaluated using the real-time PCR technique.
Findings: The frequencies of smeZ, speI, and speH genes in 12 S. pyogenes isolates were 25, 8.3, and 8.3%, respectively. The fold change rate for smeZ gene was -1.209, indicating that this gene was decreased 1.209 folds in the treated group compared to the untreated group.
Conclusion: Bacteriocin not only reduces the number of pathogens but may also affect the metabolism of the bacteria by producing toxins. The use of new antimicrobial agents in place of previous drugs for psoriasis patients could be considered as a way to treat the disease more effectively in the future.
 

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Backgrounds: Staphylococcus aureus is one of the major causes of nosocomial infections. Biofilm formation is an important virulence factor of S. aureus, leading to its high resistance to antibiotics and evasion from host defenses. This study aimed to assess the prevalence and antimicrobial resistance profile of biofilm-producing S. aureus strains and characterize genes involved in biofilm formation.
Materials & Methods: A total of 79 S. aureus strains were isolated from 1000 clinical samples and characterized using phenotypic, biochemical, and molecular tests. The biofilm production ability of isolates was examined using the microtiter assay. Moreover, the expression of genes involved in biofilm production (psm A and psm B) was screened using real-time PCR. Finally, antibiotic susceptibility testing was done using the Kirby-Bauer method and interpreted according to the CLSI M100 standard.
Findings: Out of 79 S. aureus isolates, 43 (54.4%) isolates were strong biofilm producers, 21 (26.6%) isolates were weak biofilm producers, and 15 (19%) isolates were non-adhesive. The results of real-time PCR showed that 55 (86%), 60 (93.7%), and 46 (58.2%) isolates were positive for psm A, psm B, and both genes, respectively. The results of antibiotic susceptibility testing showed that all the isolates were resistant to two or more antibiotics.
Conclusion: The high prevalence of biofilm-forming S. aureus strains in hospital environments could be a major health challenge with serious outcomes for hospitalized patients. Thus, it is necessary to disinfect hospital environments to reduce the risk of infection and spread of these microorganisms.
 

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Objective: Alpha-thalassemia is one of the most prevalent hemoglobin disorders in the world and it is a common hereditary condition caused by deletion of one or more α-globin genes. Common α-thalassemia deletions like 3.7 kb, 4.2 kb, 20.5 kb and Med can be detected by Multiplex PCR. There are, however, some unknown deletions that can not be detected by the mentioned method or even by direct DNA sequencing. In the present study, Real-time PCR was used to determine the presence or absence of unknown deletions. Materials and Methods: Real-time PCR was performed using intercalating dye SYBR Green I and α1, α2 and CLCN7 genes were amplified. Data analysis was conducted using comparative threshold method (ΔΔCT) for determination of Gene dosage of α1-globin and α2-globin genes. Results: The results showed the ratio of 0.90±0.16 for normal individuals and the ratio of 0.32±0.15 for carrier samples with deletions. In addition, Melting curve analysis confirmed the specific amplification of target genes. Conclusion: The Real-time PCR assay is simple, rapid, and reliable. It can be applied for direct determination of unknown deletions in Alpha-thalassemia carriers.

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Background: In thyroiditis, the cellular immune response plays a crucial role in triggering the production of autoantibodies. Toxoplasma gondii elicits a strong innate and adaptive immune response within the host organism. This study aims to assess the seroepidemiological prevalence of toxoplasmosis and the levels of interferon-gamma (IFN-γ) in patients with autoimmune thyroiditis (AITD) in Iraq.
Methods: This case-control survey was conducted on 100 patients diagnosed with AITD and 70 healthy individuals (non-AITD) who attended general hospitals in Thi-Qar Province, Iraq, between July and November 2023. The prevalence of anti-Toxoplasma gondii antibodies was evaluated using enzyme-linked immunosorbent assay (ELISA) kits. Serum levels of IFN-γ were measured using ELISA kits, while the expression levels of IFN-γ were assessed using real-time polymerase chain reaction (PCR).
Findings: Among the participants, 33 patients (33.00%) in the AITD group and 9 patients (12.85%) in the non-AITD group tested positive for T. gondii IgG antibodies (p < 0.001). Additionally, 2.00% of AITD patients and 1.40% of non-AITD patients were positive for anti-Toxoplasma IgM antibodies. PCR analysis revealed the presence of T. gondii parasites in 2.00% of AITD patients and 1.4% of non-AITD patients. In AITD patients with T. gondii antibodies, both serum levels and gene expression of IFN-γ were significantly elevated compared to AITD patients without T. gondii antibodies (p < 0.05).
Conclusion: Current findings suggest that individuals infected with T. gondii may experience direct effects on the thyroid gland due to elevated levels of IFN-γ. However, further analyses are necessary to validate these results.

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Objective: In this study, the possibility of prenatal diagnosis of Down syndrome with Real-Time PCR method was evaluated. In this context, optimization of a suitable method for purification of high quality DNA from amniotic fluid samples was also considered. Materials and Methods: Pregnant women who had the high risk of having babies with Down syndrome were selected according to the biochemical and sonographic data and referred to the amniocentesis center. The DNA of total 59 amniotic fluid samples were extracted with different methods including boiling method, salting out method, Procedures of DNA extraction from Blood and Cell Culture by DNPTM Kit (CinnaGen), Procedure of DNA extraction from cells by DNA Isolation Kit for cells and tissues (Roche), Procedure of DNA extraction from Tissue by MagNa Pure DNA Isolation kit (Roche), and QIAamp DNA Micro Kit (Qiagen). Then, the quality and quantity of the extracted DNA were evaluated by the NanoDrop® ND- 1000 spectrophotometer device. Real-Time PCR reaction using fluorescent dye SYBR Green I (Applied Biosystems, UK) was performed to specifically amplify DSCAM and DYRK1A2 genes and the reference gene (PMP22). Data analysis was performed using comparative cycle threshold method for the determination of the gene dosage and determining the number of copies of chromosome 21. Results: This study showed that DNA extracted from amniotic fluid samples using QIAamp DNA Micro Kit (Qiagen) has the desirable quantity and quality for Real-Time PCR. Specific proliferation of targets and reference genes was achieved and difference between normal and affected groups based on differences between their gene dosages was determined. Conclusion: Prenatal diagnosis of Down syndrome is feasible by the Real-Time PCR method using DNA samples from amniotic fluid cells extracted by QIAamp DNA Micro Kit (Qiagen). The results are comparable to the corresponding results from conventional cytogenetic methods.

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Objective: Prostate cancer is one of the most common cancer in the developed countries. Most of cancer deaths are due to development of metastasis. Hence, prevention of metastasis is critical. Silibinin is a flavonoid component that inhibits cell proliferation and causes cell death of human prostate cancer. In this study, the expression of CD82 gene in PC-3 cells treated with escalating concentrations of silibinin was evaluated which can result in new view for prostate cancer therapy. Materials and Methods: In this study, PC-3 cells were treated with different concentrations of silibinin for 24h. The LD50 was determined. RNA was extracted by trizol, then cDNA was synthesized. Precise primers were designed for CD82 and GAPDH genes by specific software. Quantity of CD82 gene expression compare to GAPDH gene in different concentrations of silibilin was analyzed using very sensitive quantitative Real-time PCR. Results: CD82 gene expression in PC-3 cells treated with 100, 150 and 200μg/ml of silibinin at 24h was increased by 1.97±0.26 (P<0.05), 3.00±0.26 and 3.43±0.43 (P<0.01), respectively. Conclusion: The results of quantitative Real-time PCR indicated that silibinin can probably decrease metastasis, by up-regulation of CD82 metastasis suppressor gene in PC-3 cells.

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Objective: In this study quantitative expression of MDR1 and hOCT1 genes in CML patients and normal people were measured using Real-Time PCR. Materials and Methods: To study quantitative expression of these genes by real-time PCR, master-mix with syber green was used. Peripheral blood samples from 30 CML patients and 27 normal persons were harvested. Real-time PCR results were analyzed with relative quantification method. Result: This study showed that in the patients group who were under treatment with Imatib, MDR1 gene expression was increased which was statistically significant. This increase has a direct relation with disease progress. Gene expression in AP and BP patients was also higher than CP patients. In contrast, hOCT1 expression in patients group in comparison with normal group was not statistically significant. Conclusion: MDR1 increase in leukemic cell membrane results in the reduction of intra-cellular drug concentration. Thus, optimal concentration of drug for inhibition of BCR-ABL tyrosine kinase is not achieved which culminated in disease progression to AP and BP phases. Moreover changes in hOCT1 gene expression as an influx transporter of Imatib could affect intracellular concentration of drug and finally determine therapy outcome. However, in this study hOCT1 gene expression was variable and was not statistically significant.

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Objective: Traditional methods for storing microbial DNA samples use the low-temperatures of 4 °C, -20°C or -80 °C. The DBC Card is a new method of storing microbial DNA at room temperature. This study evaluates the stability and determine the best storing method of Bacterial DNA on the DBC card. Methods: In this study, we used a pair of primers from the conserved domain of 16srRNA designed to identify Escherichia coli. Four different types of samples we prepared: i) bacterial suspension, ii) bacterial DNA extracted by the phenol – chloroform method, iii) bacterial lysate with lysis buffer and iv) bacterial DNA produced by the boiling method. All four samples were spotted on separate DBC cards and dried at room temperature. After periods of 3, 5 and 7 months, Escherichia coli samples were checked for DNA stability with two molecular techniques, conventional PCR with 1, 2 and 3 disks as a source of bacterial DNA (1 mm diameter) and Real-Time PCR. Results: Data showed that DNA stability was maintained after 7 months on a DBC disk, even using only one disk as a DNA source. The bacterial suspension was the best method for long-term storage of Escherichia coli DNA on the DBC Card. Conclusion: In traditional methods for storing sample, DNA quality reduces after freezing and thawing. However in the DBC method, DNA quality was maintained for a long duration. Advantages of the DBC method are easy sample handing, low cost, faster extraction, and reduced individual and environmental contamination.

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Gastric cancer is one of the most common cancers in the world. Its treatments are costly and can cause severe side effects. As a result, treatments with natural compounds, well-established therapeutics, or combinations of both groups may be effective alternatives. p-Coumaric acid (pCA) and metformin (Met) are among such anticancer treatments. Epithelial-mesenchymal transition (EMT) is a multi-purpose process that plays a critical role in gastric cancer. This process involves a complex network of biological markers participating in gastric cancer initiation and metastasis. Subsequently, the agents downregulating the expression of EMT markers may be potential anti-gastric cancer therapeutics. Because the effects of pCA, Met, and their combination on the expression of EMT markers ZEB1, Snail2, Vimentin, and VEGFA have not been inspected, the present study aimed at assessing these effects. MTT assay determined the cytotoxicity of pCA and Met on the AGS cells for 48 hours. Real-time PCR was used to evaluate the changes in the expression levels of these EMT genes after 48 hours. A combination of pCA and Met downregulated the expression of ZEB1 and Vimentin genes at low, non-cytotoxic concentrations. Therefore, they may be potential candidates for further investigations in fighting against gastric cancer.
 

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Objectives: Human cytomegalovirus (CMV) is a major life-threatening pathogen for hematopoietic stem cell transplant recipients. Specific tests are used for the diagnosis and monitoring of CMV infection in transplant patients. This study evaluates the performance of pp65 antigenemia and qualitative PCR assays for monitoring CMV in such patients. Methods: We analyzed 179 clinical samples from 41 patients by using a validated home-brewed qualitative PCR and a commercial antigenemia assay. The obtained results were evaluated using quantitative real-time PCR as the gold standard. Results: CMV was observed in 26.8% of samples analyzed by the antigenemia assay and in 42.6% of the samples by qualitative PCR. Among 179 clinical samples, 50.8% were negative and 21.2% were positive by both assays. On the other hand, 26.3% were only positive by qualitative PCR whereas 1.7% were positive by the antigenemia assay. A comparison of the results with real-time PCR showed that qualitative PCR has a higher sensitivity than the antigenemia assay (98.7% vs. 45.7%). The specificity of both assays was equal (96.8%). Quantitative results of the antigenemia assay showed good correlation with real-time PCR (r=0.715; p<0.001). Conclusion: Both the qualitative PCR and antigenemia assays have special deficiencies for efficient diagnosis of CMV infection. Therefore, effective management of CMV infection in transplant patients requires the use of other sensitive quantitative methods such as qPCR.

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Objective: Hepatitis C virus (HCV) is considered to be a worldwide health problem. In most cases, HCV infection becomes chronic and may proceed to fibrosis, cirrhosis, and hepatocellular carcinoma. Many pathological effects in cells may occur by viral proteins. The purpose of this study is to evaluate the effect of the HCV core protein on cells to induction of the fibrogenesis process. Methods: We use the LX-2 cell line that originated from hepatic stellate cells. Plasmid which expressed HCV core protein was transfected to the cells. After 72 h, RNA was extracted and treated with DNase, followed by synthesis of cDNA. Positive control cells were treated with the leptin fibrotic hormone. We used real-time PCR to measure and statistically analyze α-SMA gene expression. Results: The HCV core protein significantly increased α-SMA gene expression (p<0.05). There was more α-SMA gene expression in cells treated with leptin compared to cells treated with the HCV core protein. Conclusion: HCV infection is an impressive factor in the development of chronic hepatitis to hepatic fibrogenesis. The HCV core protein can induce a fibrogenesis process in HCV infection.  

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Corn and soybeans have the largest area of ​​GM crops in the world. Milk powder and baby food are processed foods that contain corn and soybeans. Therefore, tracking transgenic corn and soy in processed food is one of the research problems. First, 40 samples of baby food and milk powder were collected from pharmacies and supermarkets in Tehran. All the samples were extracted using Azmaelixir DNA kit, and the nucleic acid concentration was checked with a nanodrop device, and internal control genes for soy (Lectin) and corn (Zein) were checked to ensure the extraction. Then, the presence of CaMV35S transgenic gene was checked by Real-time PCR technique. The results showed the presence of transgenic genes in 2.5% of baby food samples and 10% of samples in infant formula. Therefore, in this article, the amount of transgenic gene penetration in baby food and infant formula has been investigated.
 

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The common cutworm, Agrotis segetum, is a serious soil pest of many vegetable and field crops all over the world. Morphological identification of Agrotis species is predominantly performed on adults due to the deficiency of adequate identification keys for immature stages. In international trade, the immature life stages are frequently being intercepted at point of inspection, challenging the possibilities of morphological identification. To realize a rapid and reliable identification for all stages of A. segetum, a TaqMan real-time Polymerase Chain Reaction (PCR) was developed based on the mitochondrial Cytochrome Oxidase I (COI) gene. All specimens of A. segetum (including various life stages) were detected and no cross-reactivity was observed with 5 non-target Agrotis species in the specificity tests. The tests showed to be repeatable, reproducible, and robust. The assay performed equally well with crushed insects and purified DNA, so, the efficiency was added by removing DNA extraction step. The method has proven to be suitable tools for routine identification of all life stages of A. segetum considering the speed, specificity, as well as sensitivity of the assay.

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 Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is one of the most important diseases of wheat with devastating epidemics in Iran and the world. In this study, we evaluated some Iranian wheat landraces in a greenhouse at the seedling stage against a new pathotype related to Ug99 of Pgt, which was collected from Iran and designated as TTSSK. Marker analysis was done on resistant landraces. Molecular markers for detecting some Sr genes were used. The results showed that Sr22, Sr35 and SrWeb provided resistance against TTSSK in the resistant landraces. In addition, some of the susceptible landraces that were resistant at adult stage were used for Sr2 analysis. The results showed that some of these landraces were carrying other adult plant resistance gene/genes except Sr2. To evaluate the defence gene expression in compatible and incompatible interactions, cv. Morocco (susceptible) and KC-440 landrace (resistant) were used. Sampling was done at 0, 12, 18, 24, and 72 hours post inoculation (hpi) with stem rust isolate and water as mock treatment. β-1,3 glucanase gene expressions were studied using qGLU-S and qGLU-AS primers. Also, 18srRNA, β-tubulin and EF1-α genes were used as internal control. The results showed that in incompatible interactions, the defence gene expression was increased at 24 hpi, but in compatible interactions, expression level reached the peak at 12 hpi and it significantly decreased at 18 hpi. The results revealed that the expression of defence genes such as β-1,3 glucanase was earlier in compatible interactions than in incompatible interactions, but the quantity of expressed gene was less than in incompatible interactions.

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Since the commercialization of transgenic crops in 1996, the biotech crop planted area has continuously increased. The European consumers have particularly been sceptical about transgenes in food products and EU (European Union) has enacted very complex legislation. The area of food analytics requires continuous development and improvement of detection methods to track the legislative framework and respond to consumers requirements. In the last decade, real-time PCR (polymerase chain reaction) based methods have been the methods of choice for numerous laboratories, but for various reasons, end-point PCR based methods have still been used. In our research, 73 samples of food and feed were analysed for the presence of common elements of transgene construct – Cauliflower Mosaic Virus 35S promoter (P-35S) and Agrobacterium tumefaciens Nopaline Synthase Terminator (T-NOS), using end-point PCR based methods. These samples had been previously tested for the presence of the same elements using validated real-time PCR based methods. Comparison of the used methods sensitivity showed that real-time PCR based methods have undeniable advantage. More important factor is specificity, and the fact that the list of approved Genetically Modified Organisms (GMOs) is constantly increasing necessitates updating of validation methods procedures. Considering upward trend of approved GMOs, it is important to pay more attention to the improvement and specialization of GMO detection methods.