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Recently, members of the semaphorin family have received major attention in various medical fields, especially autoimmunity. In this study, we selected semaphorin-3A (Sema3A), semaphorin-7A (Sema7A), and their receptors to determine the possible relationship between these molecules and multiple sclerosis (MS).We measured the gene expression of Sema3A, Sema7A, neuropilin-1 (NP-1), plexin-C1, and β1 integrin in the blood samples of relapsing-remitting multiple sclerosis (RRMS) patients, treated with high-dose interferon-β1a (IFN-β1a), low-dose IFN-β1a, IFN-β1b, and glatiramer acetate (GA) via quantitative real-time polymerase chain reaction (qRT-PCR) assay, and then, compared the results of treatment-naive patients with the healthy controls.The gene expression of Sema3A (P = 0.02), NP-1 (P 0.001), and plexin-C1 (P 0.01) significantly decreased in the treatment-naive group, compared to the healthy controls. Sema3A significantly increased in all treated patients, compared to the treatment-naive patients (P 0.001). However, expression of NP-1 (P 0.001), plexin-C1 (P 0.001), and β1 integrin (P 0.05) only increased in patients receiving high-dose IFN-β1a, IFN-β1b, and GA. Expression of Sema7A increased in only two groups of patients treated with IFN-β1b (P 0.001) and GA (P = 0.018), without any significant decrease in the treatment-naive group, compared to the healthy controls (P 0.05).Our findings confirm that the presence of Sema3A, Sema7A, and their receptors can play critical roles in the treatment of MS patients. Therefore, they can be potential target molecules for MS treatment in the future.

MicroRNAs (miRNAs) are epigenetic regulators of the gene expression and act through posttranslational modification. They bind to 3'-UTR of target mRNAs to inhibit translation or increase the degradation mRNA in many tissues. Any alteration in the level of miRNA expression in many human diseases indicates their involvement in the pathogenesis of many diseases. On the other hand, the regulation of the signaling pathways is necessary for the maintenance of natural and physiological characteristics of any cell. It is worth mentioning that dysfunction of the signaling pathways manifests itself as a disorder or disease. The significant evidence report that miRNAs regulate the several signaling pathways in many diseases. Base on previous studies, miRNAs can be used for therapeutic or diagnostic purposes. According to the important role of miRNAs on the cell signaling pathways, this article reviews miRNAs involvement in incidence of diseases by changing signaling pathways.

Introduction: Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease which involves the central nervous ­system. Although the primary cause of MS is obscure, effects of some cytokine and chemokine patterns in both innate and adaptive immune systems have been described. ­Objectives: Since limited studies have examined the role of interleukin (IL)-11 and chemokine CCL27 in MS, we aimed to identify changes in IL-11 and CCL27 gene expression and serum levels in relapsing-remitting MS (RRMS) patients, treated with interferon (IFN)-β and glatiramer acetate (GA). Methods: The serum level and gene expression of IL-11 and CCL27 were measured and compared between treatment-naïve MS patients and RRMS patients who were treated with high-dose IFN-β1a, low-dose IFN-β1a, IFN-β1b, and GA via enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction. Results: A significant decrease was observed in the serum level of CCL27 in treatment-naïve patients and IFN-β1b-treated patients compared to the healthy controls. On the other hand, a significant increase was found in the protein level of CCL27 in low-dose and high-dose IFN-β1a groups compared to the treatment-naïve group. In addition, CCL27 gene expression was higher in patients treated with GA than in the treatment-naïve group. There were no significant changes in the gene expression or protein level of IL-11 in all experimental groups. Additionally, a positive correlation was found between IL-11 and CCL-27. Conclusion: Our results suggest the inflammatory role of CCL27 in MS patients, while IFN-β1a seems to play a compensatory role for this chemokine.

Hydatidosisis a parasitic disease caused by the larval stage of Echinococcus granulosus with different genotypes, and major complications in vital organs such as liver, lungs and, brain. Also, this parasite can infect animals and cause economic damages. Recently, some investigations indicated that the genetic variation of the parasite affects the antigenic, immunogenic and pathogenic features. Therefore, present study conducted to genotyping of the E. granulosus larva based on mitochondrial cox1 gene in livestock in the endemic areas of Markazi province, Iran. In this study, 49 hydatid cysts samples collected from 36 sheep, 11 goats and 2 cattle from different slaughterhouses of Markazi province in central part of Iran, 2017. The mitochondrial cox1 gene was amplified and genotyping were accomplished using sequence analysis. The sequencing analysis indicated that the main genotype G1 (61%) and G3 (37%) were identified. Also, one of the samples shows similarity with the G2 (2%) genotype. The results showed the statistically significant differences between the genotypes in different livestock (P

Background: Helicobacter pylori is the main cause of stomach ulcers and gastric cancer. Hence, the diagnosis, treatment, and prevention of H. pylori infection can considerably reduce the fatality. Objectives: This study aimed to construct a dual-antigen protein by combining the antigenic regions of UreB and FlaA of H. pylori and determine its antigenicity as a promising vaccine and serodiagnosis candidate. Methods: The antigenic regions of FlaA and UreB were detected by immunological bioinformatics, amplified and joined together by polymerase chain reaction (PCR) with special primers containing linker sequences. Then, it was cloned into pET-32a and after expression and purification of the recombinant multi-epitope protein (rFlaA-UreB), its antigenicity was evaluated by immunoblotting using the sera of infected patients. Results: DNA sequencing and enzyme digestion analysis showed the rFlaA-UreB gene was successfully inserted into pET32a. The recombinant protein was produced and purified via affinity chromatography and its molecular weight was similar to what had been predicted. Moreover, data indicated that rFlaA-UreB was recognized by all patients’ sera and its sensitivity and specificity were high. Conclusions: Although the developed recombinant multi-epitope protein was very smaller and lighter than the natural forms of these two critical antigens, they all had close antigenic properties. Therefore, this recombinant protein can be an important antigen in the diagnosis and vaccination against H. pylori.

Aim: The human parainfluenza viruses (HPIVs) are common viral causes of both upper and lower respiratory tract infections in infants and children. The purpose of this study was to develop a real-time reverse transcription PCR assay to detect HPIVs in clinical specimens in a single test. Specimens: Specific primers and probes matched to conserved region of hemagglutinin–neuraminidase genes were designed. Clinical specimens were collected from hospitalized children of ≤5 years old. Results & conclusion: Of 230 specimens, 48 samples contained one of HPIV types 1–4. The most percentage of positive HPIVs belonged to HPIV2. HPIVs were found in fall, winter and spring but not in summer months. In conclusion, our in-house multiplex real-time reverse transcription PCR is a suitable technique for simultaneous detection of HPIVs in clinical samples.

Accurate and timely diagnosis of acute bacterial meningitis is critical for antimicrobial treatment of patients. Although PCR-based methods have been widely used for the diagnosis of acute meningitis caused by bacterial pathogens, the main disadvantage of these methods is their high cost. This disadvantage has hampered the widespread use of molecular assays in many developing countries. The application of multiplex assays and "in-house" protocols are two main approaches that can reduce the overall cost of a molecular test. In the present study, an internally controlled tetraplex-PCR was developed and validated for the specific detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in cerebrospinal fluid (CSF) samples. The analysis of a panel of other human pathogens showed no cross-reactivity in the assay. The analytical sensitivity of the in-house assay was 792.3 copies/ml, when all three bacteria were presentin the specimens. This value was calculated as 444.5, 283.7, 127.8 copies/ml when only S. pneumoniae, N. meningitidis and H. influenzae, respectively, were present. To demonstrate the diagnostic performance of the assay, a total of 150 archival CSF samples were tested and compared with a commercial multiplex real-time PCR kit. A diagnostic sensitivity of 92.8% and a specificity of 95.1% were determined for the present tetraplex-PCR assay. The results indicate that the established method is sensitive, specific and cost-effective, and can be used particularly in situations where the high cost of commercial kits prevents the use of molecular methods for the diagnosis of bacterial meningitis.

Accurate identification of dermatophyte species is important both for epidemiological studies and for implementing antifungal treatment strategies. Although nucleic acid amplification-based assays have several advantages over conventional mycological methods, a major disadvantage is their high cost. The aim of this study was to develop a rapid and accurate real-time PCR-based high-resolution melting (HRM) assay for differentiation of the most common dermatophyte species. The oligonucleotide primers were designed to amplify highly conserved regions of the dermatophyte ribosomal DNA. Analysis of a panel containing potentially interfering fungi demonstrated no cross reactivity with the assay. To evaluate the performance characteristics of the method, a total of 250 clinical isolates were tested in comparison with the long-established PCR-RFLP method and the results were reassessed using DNA sequencing, as the reference standard method. The assay is able to type dermatophytes using normalised melting peak, difference plot analysis or electrophoresis on agarose gel methods. The results showed that, in comparison to PCR-RFLP, the developed HRM assay was able to differentiate at least 10 common dermatophytes species with a higher speed, throughput and accuracy. These results indicate that the HRM assay will be a useful sensitive, high throughput and cost-effective method for differentiating the most common dermatophyte species.

MicroRNAs play an important role in neuronal development and function. miR-124 is the most abundantly expressed miRNA in the nervous system. Several different mRNA targets have been proposed for miR-124, but the precise function of endogenous miR-124 and its mRNA targets remain to be further elucidated. Specificity protein 1 (Sp1) is a transcription factor that plays key roles in many cell processes including cell cycle. However, this transcription factor is nearly absent in differentiated neurons and a remarkable suppression of Sp1 expression was shown after neurogenesis. Since miR-124 is expressed abundantly in neurons and because Sp1 levels decrease during neurogenesis, it is possible that miR-124 could regulate the expression of Sp1 during neuronal development. Therefore, the aim of the present study was to evaluate the putative targeting of Sp1 by miR-124. Overexpression of miR-124 using a plasmid coding for pri-miR-124 in HEK293 cells decreased the expression of Sp1 mRNA. The results of dual-luciferase reporter assay demonstrated that miR-124 directly targeted the 3′-untranslated regions of Sp1 mRNA. To evaluate whether Sp1 expression was regulated by miR-124 during the process of neuronal differentiation, Adipose-derived mesenchymal stem cells (A-MSCs) were differentiated into neuron-like cells. The results of qPCR analysis showed that with the gradual increase of miR-124 expression during neurogenesis, the expression of Sp1 mRNA decreased accordingly. In summary, this study demonstrated for the first time that miR-124 is able to suppress Sp1 expression, which in turn affected the neuronal differentiation of mesenchymal stem cells. J. Cell. Biochem. 116: 943–953, 2015. © 2015 Wiley Periodicals, Inc.

OBJECTIVES: Incidence of methicillin-resistant Staphylococcus aureus (MRSA) is increasing every year, especially in burn patients with a high rate of morbidity and mortality. Molecular and epidemiologic studies are useful practices for understanding the relatedness of isolates in a single patient or a hospital. This study aimed at determining molecular characterizations of isolates collected in 2006 and 2014 using S. aureus-specific staphylococcal protein A (Spa) typing and Multilocus Sequence Typing (MLST) methods. MATERIALS AND METHODS: Totally, 71 MRSA isolates were collected during the last two studies (2006 and 2014) from burn patients at Taleghani Burn Centre. After confirmation, all isolates were analysed using MLST and Spa typing methods. RESULTS: We reported the emergence of Spa type t021, ST-30-IV MRSA isolates, which were PVL-positive in 14.6% of the cases and t12366, ST-8-IV isolates, which were PVL-negative in 9.8% of the cases. In 2014 study, Spa typing of MRSA isolates revealed five different spa types. Overall, in two studies, t037, ST-239, SCCmec III, and CC8 were predominant clones and they were reported in 63% of the cases. CONCLUSION: The predominance of ST-239 in this region during the last eight years is a major concern. It also has a disturbing impact on the management of staphylococcal infections. Moreover, the SCCmec type IV strain is able to disseminate rapidly in hospital environments, demanding an improvement in infection-control policy

Background: Helicobacter pylori is a common cause of chronic infections worldwide. Helicobacter pylori urease is a multimeric enzyme with a molecular weight of 530 kDa, which is collected from two separate subunits of UreA (26.5 kDa) and UreB (61.7 kDa). Objectives: This study aimed to evaluate the antigenic properties of UreB recombinant protein as a vaccine candidate for the infected sera of humans and mice. Methods: In this study, the highly antigenic region of UreB gene (609 bp) was detected, using immunological bioinformatics for epitope mapping, amplified by polymerase chain reaction (PCR). Afterwards, the antigen was cloned, using a pBSK cloning vector and was inserted into a pET-32a expression vector; the target protein was then expressed and purified. Eventually, antigenicity was examined by immunoblotting on the sera of humans infected with H. pylori UreB recombinant protein. Results: The sequencing results of PCR were indicative of UreB gene cloning into the recombinant plasmid. Production of the protein was induced by isopropyl β-D thiogalactopyranoside (IPTG), and the expressed protein was purified via dialysis, using the Ni-NTA kit. In addition, the recombinant protein with a molecular weight of 42 kDa was recognized by antibodies in Western blotting. Conclusions: The evaluation of antibodies was indicative of high antigenic properties for the immunogenic fragment, predicted by immunological bioinformatics. Therefore, UreB recombinant protein might be a proper antigen for the development of vaccines against H. pylori and other diagnostic kits.

Introduction Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer. One of the signal transduction pathways related to NSCLC is Unfolded Protein Response (UPR), which is mainly regulated by GRP78 ( HSPA5 , Gene ID: 3309). The aim of this study was to employ bioinformatics tools to predict microRNAs (miRNAs) affecting GRP78 expression, experimentally validate interaction of these miRNAs with GRP78 and also evaluating the expression correlation of GRP78 and its predicted miRNAs in clinical samples. Materials and methods Various software were used to predict miRNAs that simultaneously target all upstream and downstream components of GRP78 in the UPR, as well as the main components of PI3K/AKT, MAPK, ErbB and calcium pathways. For experimental analysis, 36 pairs of Formalin-Fixed Paraffin-Embedded (FFPE) lung tumor and non-tumor tissue samples were obtained. Additionally, A549 and QU-DB lung cancer cell lines were used for expression determination of GRP78 and its predicted targeting miRNAs. We also employed a luciferase assay to evaluate interactions between candidate miRNAs with the 3′-UTR of GRP78. Results hsa-miR-495 and hsa-miR-199-5p were chosen based on several criteria including thermodynamic binding features of miRNAs to the target transcripts, number of recognition sites, and conservation of binding sites within the 3′-UTR of GRP78. RT-qPCR data revealed a significant up-regulation of GRP78 (3.87 times, P = 0.002) and down-regulation of miR-199a-5p (0.13 times, P = 0.0001) and miR-495 (0.085 times, P = 0.0001) in tumor samples. Luciferase assay confirmed an interaction of hsa-miR-199a-5p and hsa-miR-495 with the 3′-UTR of GRP78 transcript. In addition, over-expression and competitive inhibition of the aforementioned miRNAs, significantly altered the expression of GRP78 and spliced XBP1 level. Conclusion Our data revealed a significant up-regulation of GRP78 and a concomitant down-regulation of miR-495 and miR-199a-5p in NSCLC. Accordingly, our data suggest a causative role for miR-199-5p and miR-495 in tumorgenesis of lung and probably other cancer types.

Characterization of clinically important dermatophytes in North of Iran using PCR-RFLP on ITS region. Didehdar M1, Shokohi T2, Khansarinejad B3, Ali Asghar Sefidgar S4, Abastabar M5, Haghani I5, Amirrajab N5, Mondanizadeh M6. Abstract OBJECTIVE: Dermatophytosis is considered as one of the major public health problems in the world. Accurate identification of the dermatophytes species is important for recognition of the environment and animal sources of infection. In this survey, 1500 clinical specimens taken from outpatients suspected of dermatophytosis and they were examined by direct microscopy and culture. MATERIALS AND METHODS: In total, three hundred and twelve dermatophytes were isolates recovered in culture, they were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method based on the ribosomal DNA internal transcribed spacer (ITS) regions. RESULTS: Tinea pedis was the most common clinical form (32.1%) followed by tinea cruris (24.4%), tinea manuum (16.4%), tinea unguium (8.3%), tinea corporis (7.3%), tinea faciei (6.4%), and tinea capitis (5.1%). Trichophyton interdigitale was the most frequent isolate (38.2%), followed by Trichophyton rubrum (29.8%), Epidermophyton floccosum (16.6%), Trichophyton tonsurans (14.8%) and Microsporum canis (0.6%). The frequency of dermatophytosis was higher in males than in females and in the age-group of 21-30 years. CONCLUSION: Our finding indicated that the incidence of dermatophytosis caused by anthropophilic dermatophytes in Mazandaran province is increasing. Also, this study provides valuable data for the prevention and control of dermatophytosis in the southern coast of the Caspian Sea. KEYWORDS: Dermatophytes; Identification moleculaire; Molecular identification; Nord de l’Iran; North of Iran

Background: The Crimean-Congo hemorrhagic fever (CCHF) virus causes severe disease in humans, with a high mortality rate. Since, there is no approved vaccine or specific treatment for CCHF, an early and accurate diagnosis, as well as reliable surveillance, is essential for case management and patient improvement. Objectives: For this research, our aim was to evaluate the application of a novel SYBR Green based one-step real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay for the in-house diagnosis of the CCHF virus. Patients and Methods: In this experimental study, the highly conserved S-region sequence of the CCHF viral genome was first adapted from GenBank, and the specific primers targeting this region were designed. Then, the viral RNA was extracted from 75 serum samples from different patients in eastern Iran. The sensitivity and specificity of the primers were also evaluated in positive serum samples previously confirmed to have the CCHF virus, by this one-step rRT-PCR assay, as well as a DNA sequencing analysis. Results: From a total of 75 suspected serum samples, 42 were confirmed to be positive for CCHF virus, with no false-positives detected by the sequencing results. After 40 amplification cycles, the melting curve analysis revealed a mean melting temperature (Tm) of 86.5 ± 0.6°C (quite different from those of the primer-dimers), and the positive samples showed only a small variation in the parameters. In all of the positive samples, the predicted length of 420 bp was confirmed by electrophoresis. Moreover, the sensitivity test showed that this assay can detect less than 20 copies of viral RNA per reaction. Conclusions: This study showed that this novel one-step rRT-PCR assay is a rapid, reliable, repeatable, specific, sensitive, and simple tool for the detection of the CCHF virus.

Background: Helicobacter pylori (H.pylori) is one of the most common chronic infections worldwide. The H. pylori Urease is a high molecular mass (530 kDa) multimeric enzyme collected of two separate subunits, UreA (26.5 kDa) and UreB (61.7 kDa). Recently, many researchers are working in the expansion of a vaccine to prevent H. pylori infection, and of the various candidate antigens, the majority promising is the B subunit of the Urease protein (Urease B), for the reason that immunization of mice with purified Urease B has resulted in better immunogenicity and protection as contrast to the use of Urease A. The aim of this study was to high level expression and evaluation of antigenic property of recombinant UreB protein with infected human and mice sera as a vaccine candidate. Materials and Methods: In this experimental study, the highly antigenic region of UreB gene (609 base pair) was detected by best immunobioinformatics methods of epitope mapping, amplified by PCR method and was cloned into the cloning vector pBSK and then inserted into the expression vector pET-32a. The target protein was expressed and purified. Finally antigenicity was studied by western blotting using human sera infected with H. pylori UreB recombinant protein. Results: PCR and sequencing results showed the successful cloning of the target gene into the recombinant vector. The expression of protein was induced by IPTG and the expressed protein was purified with Ni-NTA kit and dialysis. The recombinant protein with molecular weight about 42 KDa was recognized by antibodies in western blotting. Conclusion: Evaluation of antibodies studies have shown that the predicted immunogenic fragment by best immunobioinformatics tools antigenic properties is high., so this Recombinant UreB protein is a good candidate for the design of H. pylori vaccine and diagnostic kits.

Background: Pseudomonas aeruginosa is an opportunistic pathogen that infects patients with cystic fibrosis, burning wounds, ophthalmic traumas and immunodeficiency. Objectives: The aim of the present study was to compare the efficiency of newly designed primer sets with some previously published primers for PCR detection of exoA, oprL and algD genes from P. aeruginosa. Materials and Methods: A total of 150 clinical specimens were inoculated into the routine and selective culture media for P. aeruginosa isolation. Specific primers were designed by bioinformatics analysis for detection of the virulence genes determinants, exoA, oprL and algD. The sequences of these three genes were obtained from NCBI and multiple alignments were performed to find the conserved sequences of each gene to design the primers. Multiple alignment and primer design steps were carried out by the AlleleID software, version 7.0. Results: Microbiological culture methods resulted 70 P. aeruginosa strains isolated from 70 of the 150 clinical specimens. The results of the PCR assay using the newly designed exoA, oprL and algD primer sets were positive in 68, 70 and 69 clinical samples which represent 97.2%, 100% and 98% sensitivity for each primer set, respectively. The PCR results using previously published exoA, oprL and algD primer sets were positive only in 57, 49 and 28 specimens that correspond to 81.5%, 70% and 40% sensitivity, respectively. Conclusions: The results of the present study showed that in comparison with previously published primer sets, P. aeruginosa infection can be diagnosed with higher sensitivity and specificity by the conventional PCR assay using the newly designed primers. It was also shown that the results of the PCR assay on clinical samples of severe infections became positive much earlier than the results of conventional culture method.

High-level Expression and Purification of Active Human FGF-2 inEscherichia coli by Codon and Culture Condition Optimization Mohammad Reza Soleyman 1 ; Mostafa Khalili 1 ; Behzad Khansarinejad 2 ; and Maryam Baazm 3, * 1 Department of Biotechnology, School of Medicine, Arak University of Medical Sciences, Arak, IR Iran 2 Department of Microbilogy and Immunology, School of Medicine, Arak University of Medical Sciences, Arak, IR Iran 3 Department of Anatomy, School of Medicine, Arak University of Medical Sciences, Arak, IR Iran *Corresponding Author: Maryam Baazm, Department of Anatomy, School of Medicine, Arak University of Medical Sciences, Arak, IR Iran. Tel: +98-9166621131, Fax: +98-8634173526, E-mail: Dr.Baazm@arakmu.ac.ir. Abstract Background: Basic fibroblast growth factor (bFGF) is a member of a highly conserved superfamily of proteins that are involved in cell proliferation, differentiation, and migration. Objectives: The objective of this study was to overexpress and purify the high-level active human bFGF in Escherichia coli (E. coli). Materials and Methods: This experimental study was conducted in the Islamic Republic of Iran. After codon optimization and gene synthesis, the optimized FGF-2 gene was subcloned into plasmid pET-32a. pET32-FGF-2 was transformed into E. coli BL21 for expression. The cultivation parameters were optimized to produce a high yield of FGF-2. Results: The optimal conditions were determined as follows: cultivation at 37°C in TB medium, with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), followed by post-induction expression for 6 h. Under the abovementioned conditions, the expression volumetric productivity of FGF-2 reached 1.48 g/L. Conclusions: A fusion tag from the pET32 expression plasmid permits the recovery of the recombinant fusion FGF-2 from E. coli,without affecting its biological activity. Keywords: Fibroblast Growth Factor 2; Codon; Protein; E. coli

Background: Toxoplasma gondii is an obligatory intracellular protozoan parasite, which infects human beings. Since the current antigens used for diagnosis or vaccination are contaminated with non parasitic material in which the parasite is grown, it is tried to produce recombinant antigens to design vaccines against toxoplasmosis, or make diagnostic kits. Choosing the type of antigen to produce recombinant vaccine or diagnostic kits is considerably important. The rhoptry protein 1 is one of the excretory-secretary antigens of Toxoplasma which seems to be an appropriate candidate in production of recombinant vaccines and diagnostic kits. Objectives: The current study aimed to produce rhoptry protein 1 from Iranian strains of Toxoplasma for further investigations. Materials and Methods: Genomic DNA was isolated from tachyzoite of parasite by phenol chloroform method and gene fragment was amplified by polymerase chain reaction. The polymerase chain reaction products were ligated into restriction enzymes sites of pTZ57R/T cloning vector. The product was sub-cloned into a prokaryotic expression plasmid (pET32a). The recombinant expression vector containing rhoptry protein 1 sequence was transformed into Escherichia coli BL21 pLysS and was induced by isopropyl β-D-1-thiogalactopyranoside. The sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting methods were used to confirm the production of protein. Results: The result showed that the desired molecular weight protein is produced. Recombinant protein was confirmed by western blot using Ni-NTA- conjugate. Conclusions: The result of this study showed that recombinant ROP1 Toxoplasma was produced successfully. In the present study, unlike other studies, the goal was to express full length of ROP1. Also this protein was produced alone not fused with other proteins.

Quantitation of human cytomegalovirus (HCMV) DNA is used to monitor immunocompromised patients in order to identify patients for preemptive therapy. Although several commercial qPCR assays are available for quantitation of HCMV, their major disadvantage is the high cost. In the present study, an internally controlled quantitative real-time PCR assay based on hydrolysis probe technology was developed for detection and quantitation of HCMV DNA in plasma samples. To demonstrate its performance characteristics, a total of 178 plasma samples from 102 kidney and hematopoietic stem cell transplanted patients were tested. The assay showed good precision and reproducibility, and an analytical sensitivity of 288.5 copies/ml or 17.6 copies/reaction. A sensitivity of 93.1% and a specificity of 96.6% were determined by examining clinical samples. Analysis of a panel containing potentially interfering viruses demonstrated no cross-reactivity with the assay. A strong correlation was observed between this qPCR method and the commercial Artus(®) CMV RG PCR kit (R=0.948; P=0.000). These results indicate that the affordable internally controlled qPCR method described will be useful for monitoring HCMV infection in plasma samples of immunocompromised patients.

INTRODUCTION: Prevalence of influenza A virus (Flu-A), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) was assessed in children with acute respiratory infections (ARIs). METHODS: Nasopharyngeal aspirates and throat swabs were subjected to real-time polymerase chain reaction (PCR) to detect RSV and Flu-A and to conventional PCR to detect hMPV. RESULTS: Of the 156 children assessed, 93 (59.6%) carried at least one virus, with 35.9% positive for RSV, 14.1% for hMPV, and 9.6% for Flu-A. The prevalence of co-infections was 2.6%. CONCLUSIONS: The high detection rate may reflect increased sensitivity of real-time PCR compared to traditional PCR and viral culture.