فهرست مطالب کیوان مجید زاده

The present study designed a loop mediated isothermal amplification (LAMP) method to rapidly detect Coxiella burnetii (C. burnetii) and develop a sensitive Real-time quantitative LAMP (Q-LAMP) assay to quantify Q fever.

Primers were specifically designed for use in targeting the com1 conserved gene of C. burnetii to carry out LAMP detection of the agent causing Q fever. After obtaining the LAMP reaction, the sensitivity of the method was assessed by preparing a serial ten-fold dilution of a plasmid carrying com1 gene.

The assay’s sensitivity for the visual detection of changes in turbidity or turbidimeter and electrophoresis of agarose gel were 0.4 fg and 0.04 fg, respectively. Hence, the lower limit of detection was 120 and 12 copies of the gene detected in 60 min. The results of this study were indicative of the simplicity, rapidness, sensitivity, and specificity of the LAMP assay for C. burnetii detection and a probable improvement of the diagnostic procedure used in clinical laboratories.

The assay specificity was assessed using Coxiella genomic DNA and a panel of Gram-negative and Gram-positive bacteria. The LAMP assay was shown to be highly specific for detection of Coxiella without any observable amplification products from non-Coxiella organisms.

 The Salmonella origin diseases is wide which could be from gastroenteritis, enteric fever, bacteraemia and focal infection. The infection of salmonella usually comes from the consumption of the contaminated food or water with animal or human excrement. This study aimed to design a Quantitative-LAMP method to real-time detection of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of Typhoid fever.

A new LAMP primer set specifically was designed based on ViaB gene and used for detection of S. Typhi. To evaluate the analytical specificity, the genome of some Salmonella-related and non-related bacteria were subjected to the S. Typhi LAMP assay. Also, the analytical sensitivity or limit of detection of the assay was evaluated. Furthermore, in the experiment, turbidity in tubes was assessed by a turbidimeter and then a standard curve was depicted by plotting time threshold values against the log of ViaB gene copy number. With this standard curve, we could use the method to make a quantitative.

The Salmonella Typhi LAMP assay specifically assessed the ViaB gene. The analytical sensitivity of the assay when using agarose gel electrophoresis or amplification plot obtained from Loop amp real-time turbidimeter system was 0.28 fg whereas with direct observation of fluorescent color change to evaluate amplification was 2.8 fg. So, the lower limit of detection of the assay when applying the revealing methods was ~1 and 8 copies of the ViaB gene respectively. 

The Salmonella Typhi LAMP assay is a simple and accurate tool to detect the causative agent of Typhoid fever that may designate to apply in clinical laboratories

HER2-enriched subtype of breast cancer has a worse prognosis than luminal subtypes. Recently, the discovery of targeted therapies in other groups of breast cancer has increased patient survival. The aim of this study was to identify genes that affect the overall survival of this group of patients based on a systems biology approach.

Gene expression data and clinical information on 58 patients with HER2-enriched cancer were downloaded from The Cancer Genome Atlas (TCGA). Co-expression modules were identified using the weighted gene co-expression network analysis (WGCNA). The Cox regression was used to determine the modules that had a significant relationship with the overall survival (OS) endpoint. Single-gene survival analysis was performed within the selected module. Finally, functional annotation to explore the significance of genes was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID).

Of the six identified co-expression modules, two had significantly poor prognoses. Single-gene survival analysis showed that 39% of genes in the selected modules were identified as significant. The genes were mainly related to the biological pathways such as Ubiquitin-mediated proteolysis and RNA degradation. CHAMP1, PPP1R26, PRRC2B, KANSL3, and ANAPC2 were identified as the 5 most important genes associated with reduced OS, in order of significance.

The systems biology approach can provide appropriate results relate to patient survival analysis. In this study, some genes were identified to be used as prognostic biomarkers in experimental studies related to the OS in the HER2-enriched subgroup. These genes can be considered potential candidates for therapeutic targets in this group of patients.

Yersinia pestis, a gram-negative rod belonging to the Enterobacteriaceae family, is the causative agent of plague. Classical methods of detecting the organisms are time-consuming, expensive and dangerous. The aim of the study was to design a Real-time PCR assay on the basis of the pla gene of Yersinia pestis. In this research the Real- time PCR test was optimized by using special primers for targeting pla gene. After preparing 10-fold serial dilutions of the pla and their analysis by the assay, the last dilution showing a fluorescent signal was confirmed as the limit of detection (LOD). A standard curve based on the Ct values was depicted, so the assay was developed to quantify the target gene. The analytical specificity was determined by subjecting the genome of some control negative bacteria to the assay. In this experiment, negative control genomes did not show detectable signals in the assay. The last dilution of pla plasmid which showed a fluorescent signal was 4.5 fg. So, the lower detectable copy numbers of the gene in a 20 μl PCR reaction was calculated as 1×103.

Mycoplasmas are one of the most serious contaminants of cellular cultures and their biological productions. Mycoplasma diagnosis is conducted on the basis of culture and molecular methods. These methods are different from each other in terms of accuracy, reliability, and sensitivity. This study aimed to trace the mycoplasma contaminations in culture samples using 16S rRNA specific primers based on the PCR method.
Methods and Materials: 16S rRNA gene sequence of cell culture contaminant mycoplasmas were extracted from gen bank and were sorted using gene sequence pattern. Specific primers were designed using obtained synonymies. To build positive control structure, 16S rRNA gene proliferated based on the PCR methods then cloned in a plasmid vector.
Electrophorus of PCR product of 16S rRNA revealed a band of 219bp length on Agarose gel. This pattern was not observed in negative control samples. Moreover, the fragment proliferation with the same size in recombinant plasmid confirmed the authenticity of cloning.
Discussion and Due to its high speed and accuracy, the method has potential for using in cell culture laboratories.

ESBL is an important factor in antibiotic resistance. Antibiotic resistance has been proposed as a global problem in recent decades and it causes many deaths all over the world; therefore, rapid and accurate diagnosis can be major step in solving this problem. This study aimed to identify SHV-type gene with PCR method.
Methods and Materials: SHV gene sequences were extracted and aligned from Genbank. Specific PCR primers were designed according to the consensus of the SHV genes. A plasmid containing positive control of the gene was created by using PCR and cloning methods. The assay specificity was evaluated using Brucella genomic DNA and a panel containing genomes of 10 gram-positive and gram-negative organisms. The PCR assay was highly specific and no amplification products were observed from the non-SHV organisms, an other test of the specifity was done by the use of CTX-M and TEM betalactamases.
A band with a the length of 199bp was indicated in the agarose gel electrophoresis of the PCR product in accordance with the desired sequence. Showing the same fragment in PCR confirmed the recombinant plasmid of coloning product containing the SHV gene. When the plasmid was subjected to the SHV-PCR, a ladder-like pattern was visualized on the agarose gel. The pattern has never observed in the case of negative controls.The results of specify assay conveyed that primers were separately for SHV gene.
Discussion and The results of this study indicated that PCR assay is a simple, rapid, sensitive and specific technique for detection of SHV gene that may improve diagnostic potential in clinical laboratories.

Calcium phosphate nanoparticles such as nano hydroxyapatite have shown excellent physical, chemical and biological properties and have allocated special place in regenerative medicine applications. Recently, the inhibitory effect of hydroxyapatite, as a calcium phosphate, on many cancers has been reported.
In this article β-tricalcium phosphate was synthesized using co-precipitation method and its physicochemical properties were studied (SEM, FTIR and XRD). Inhibitory effect of beta-tricalcium phosphate nanoparticles on the growth and proliferation of breast cancer cell line MCF-7 was investigated.
In vitro MTT assay studies showed that the inhibitory effect was dependent on the concentration of tricalcium phosphate nanoparticles. In addition, the results demonstrated that the inhibitory effect was 78% attributed to 50 mg. L-1 concentration of tricalcium phosphate nanoparticles.
In lowest concentrations, the inhibitory effect of tricalcium phosphate nanoparticles was higher than others.

Vitamin D and insulin play an important role in susceptibility to polycystic ovary syndrome (PCOS), and therefore vitamin D receptor (VDR), parathyroid hormone (PTH), and insulin receptor (INSR) gene variants might be involved in the pathogenesis of PCOS.

The present study was designed to investigate the possible associations between polymorphisms in VDR, PTH, and INSR genes and the risk of PCOS.

VDR, PTH, and INSR gene variants were genotyped in 35 women with PCOS and 35 controls using Polymerase chain reaction – Restriction fragment length polymorphism method. Furthermore, serum levels of glucose and insulin were measured in all participants.

No significant differences were observed for the VDR FokI, VDR Tru9I, VDR TaqI, PTH DraII, INSR NsiI, and INSR PmlI gene polymorphisms between the women with PCOS and controls. However, after adjustment for confounding factors, the VDR BsmI “Bb” genotype and the VDR ApaI "Aa" genotype were significantly under transmitted to the patients (p= 0.016; OR= 0.250; 95% CI= 0.081-0.769, and p= 0.017; OR= 0.260; 95% CI= 0.086-0.788, respectively). Furthermore, in the women with PCOS, insulin levels were lower in the participants with the INSR NsiI "NN" genotype compared with those with the "Nn + nn" genotypes (P= 0.045).

The results showed an association between the VDR gene BsmI and ApaI polymorphisms and PCOS risk. These data also indicated that the INSR "NN" genotype was a marker of decreased insulin in women with PCOS. Our findings, however, do not lend support to the hypothesis that PTH gene DraII variant plays a role in susceptibility to PCOS.

The great scientific Jihad that has been launched in our country by the order of the Supreme leader primarily requires a roadmap and a strategic plan. According to the significant place for science and technology in the 2025 perspective (20-years national master-plan) for the Iranian armed force (AJA), achieving the highest rank of health and the best scientific reputation in military medicine in the region have been targeted for the Iranian armed force. AJA University of Medical Sciences has made the draft of this roadmap according to the religious rules and the Imam Sadegh’s scientific model, refined and amended based on the expert opinions and will resolute the final draft to be applied by the whole military health network.

This model have been examined with methods such as future studies, strategic analysis, interviews and Delphi technique and also challenges for obtaining the goals have been discussed in the report and the roadmap toward the viewpoint has been structured. Also, a shortcut monitoring of the first phase of operational plan has been evaluated.

Based on the Analysis performed, the map principles such as core values, policies, strategies, prioritiesand plans has been structured.

It seems that the motivation by chiefs, departments, academics and personnel for implementation of Military Medicine Roadmap (MMR) is more important than the roadmap inscription itself. Therefore, we prepared a document that was signed by academics and military medicine commanders as a certified organizational pledge in order to inspire the whole system toward appropriate performance for this great Jihad.

Yersinia pestis, the causative agent of plague is a Gram negative rod belonging to family of Entrobacteriaceae. CDC has categorized the organism as a priority A biological agent because of its high morbidity and mortality and easy distribution through infective respiratory droplets. We conducted a comprehensive study for the conventional, molecular and modern diagnostic methods of the organism. Biological safety, cultivation and detection assays are the main parts of the present paper. Y.pestis can be recognized in the laboratory by bacteriologic, serologic and molecular methods. Currently, there are various homemade and commercially molecular methods to apply in the clinical laboratories. Culture based methods to detect Yersinia pestis are laborious and time consuming. The molecular methods are rapid, accurate and reliable, so they have high potential to apply in clinical laboratories.

Keeping various species of reptiles as pets has become popular in Iran alongside other parts of the world. On the other hand, Salmonellosis is one of the most important zoonotic diseases and reptiles have been known as reservoirs of Salmonella. Therefore, this study was designed to assess Salmonella infection in reptile pets of Tehran. Fecal samples were collected and cultured for Salmonella isolation from 270 reptiles referred to the specialized veterinary clinics in Tehran. Statistical analysis was conducted on the data. Salmonella was cultivated from 142 samples (52.6%). Salmonella isolates belonged to a variety of serogroups; however, more than half of them belonged to serogroups B and C. Most tested reptiles were healthy and most owners were unaware of the risk of Salmonella. Possible contact of these animals with immune-compromised people was recorded in many cases. The results of this study revealed that considering the fact that reptile pets are becoming more popular in Iran, educating reptile owners who are mostly unaware of the reptile’s safe keeping methods is a necessity. Finally, more studies are suggested to further investigate the role of reptiles in the epidemiology of human salmonellosis in Iran.

Q-fever in humans and coxiellosis in animals. This bacterium can survive in the environment out of the Coxiella burnetii is the etiologicagent of a zoonotic disease which named specific host. Accordingly، it categorized by the CDC in bioterrorism agents ‘category B. Consequently، rapid detection of the bacteriumadministratesthe treatment of disease.

world and Iran scientific databases was conducted. This paper includes biological safety، cultivation and A review study on the different researches on laboratory diagnosis field in the detection assays.

Detection of Coxiella burnetii can be done byclassical methods (isolation، cultivation in the appropriate cell line such as P388D1، J774، and L929)، serologic tests (immunofluorescence، micro immunofluorescence، complement fixation test and ELISA) and molecular biology methods (PCR، Nested PCR and Real Time PCR).

While have existed kinds of detection methods for this agent، costly، need to specific laboratory and time consuming are the limitation of the mentioned techniques. Molecular methods due to accuracy and high rapidity in detection can be effective.

Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are considered as an important source of adult stem cells in tissue engineering and cell therapy. They are present in various tissues such as, endometrium as the supportive cells. According to anatomical position of endometrial mesenchymal stem cells that put them in neighborhood of the fetus, they may have a significant role in fetus tolerance during pregnancy. This study was conducted to evaluate the molecular mechanism of immunosuppressive affect of endometrial mesenchymal stem cells. Mesenchymal Cells from bone marrow and endometrium were cultured at density of 2 ×105 cells/ml at presence of 100IU/ml and 500IU/ml INF-γ (IFN-gamma) and expression of indoleamine 2 3-dioxygenase (IDO) were studied after 72 hours by real-time PCR and immunocytochemistry. The study showed that IDO expression in cells exposed to IFN-γ was increased compared to the cells in the absence of IFN-γ (p<0.05). Additionally, up regulation of IDO expression was higher in endometrial cells than bone marrow cells. From these results it is concluded that endometrial mesenchymal stem cells may be used as a good candidate for cell therapy.

Streptococcus pneumoniae accounts for bacterial meningitis and is an important cause of morbidity among children and elderly. Control of this disease depends on rapid detection of the causative bacteria. The methods for detection of Streptococcus pneumoniae are gram staining, culture, and serological tests. These tests are time consuming and are limited by antimicrobial agents leading to false negative results. Currently, molecular methods such as PCR are used routinely for detection of infectious organisms. This study was performed with the aim of designing an improved PCR assay for the detection of Streptococcus pneumoniae. The specific diagnostic primers were designed based on ply gene of the bacterium. After amplifying the target gene on the genomic DNA, the PCR product was cloned in pTZ57R/T plasmid and the confirmed pTZ-ply plasmid was used as positive control in next experiments. Sensitivity of the assay was determined by performing the PCR on 10-fold serial dilutions of pTZ-ply. Specificity of the assay was determined using the genomic DNA of other related or unrelated bacterial species. The PCR, as expected, generated a 727bp amplicon. No PCR amplification was observed on the genome of negative controls. These findings indicate high specificity of the PCR. The lowest limit of detection of the assay in the detection of the ply gene was 250 copies in a 25µl reaction. The high sensitivity, specificity, and rapidity of the designed assay suggested the assay as an appropriate test for use in clinical laboratories. The further evaluation of the assay using clinical samples or artificially contaminated materials will confirm the application of this assay in clinical settings.

Avian influenza (AI) viruses have been isolated from a wide diversity of free-living avian species representing several orders. Since 1998, H9N2 AI outbreaks have been one of the major problems in Iranian poultry industry. In 2006, H5N1 was reported in swans in the north of Iran first, but until now there has been no official report from commercial flocks in Iran. The aim of this study was Molecular Surveillance of Avian Influenza in Bird Parks of Tehran, Iran. In this study, 100 fecal samples from different avian species of Public and Bird Parks (The avian species included Pigeon, Duck, Swan, Parrot, Crow and Sparrow) were collected in Tehran, in the central region of Iran during November and December 2009. RNA extraction and RT-PCR have been done according the WHO Instruction for detection of Influenza Type A. In 14% of samples genetic materials (RNA) were detected. Species including duck and sparrow were positive. This is the first report of AIV detection in this these species in Iran. Due to emergence of new H1N1 influenza and bird flu throughout the world and in regional countries, surveillance programs for monitoring the spread of these viruses need to be redesigned. Surveillance activities for AI in wild birds should be continued to provide further virological (subtype) and epidemiological (Phylogenic Study) information about circulating viruses.

Haemophilus influenza (H. influenza) is an important cause of meningitis in infants and children aged less than five years. For this reason the early diagnosis of this bacterium is important. Studies have shown that molecular methods are specific tests for early dete-ction for this agent. The purpose of this study was to design a PCR assay for the rapid detection of H. influenzae bacterium.

In this study specific primers were designed based on ompp6 gene and polymerase chain reaction (PCR) assay was setup. To create the standard positive control، the PCR product was cloned in pTZ57R/T vector. The existence of the desired gene in the T-vector was confirmed by digestion and sequencing processes. Sensitivity of the PCR assay was determined by preparing a serial tenfold dilutions of the positive control plasmid with starting concentration of 11ng/µl. The Specificity of the assay was verified by using of PCR on the genomic DNA of a variety of bacteria.

PCR results showed a band of the expected size 280bp. Sensitivity results indicated that the limit of detection of the assay was 317 copy numbers. No amplify-ication was observed after PCR in negative control bacteria genomic DNA. This outc-ome proved the specificity of the PCR assay. Discussion &

The results of this study showed that the PCR assay is a rapid، highly sensitive and specific test for detection of H. influenzae bacterium.

Neisseria Meningitidis is a causative agent of the bacterial meningitis, a life-threatening disease in children and adults. There are several classic methods for the diagnosis of this bacterium but these are time consuming and unreliable. The goal of this present study was to establish a PCR based rapid detection for Neisseria meningitidis.

In this study the target gene was crgA so the specific primers were designed for it. The PCR reaction was set up on the DNA genomic of N meningitidis. To create a positive control, the PCR product was cloned in the pTZ57R/T vector. The presence of crgA gene in the T-vector was confirmed by the digestion and sequencing. The sensitivity of this assay was tested by a serial dilution of the positive control plasmid with starting concentration of 70ng/µl. The test specificity was checked by performing the PCR on the other bacterial genomes.

This test amplified a DNA fragment with expected size of 500bp. The results of the digestion and sequencing proved the presence of crgA gene in the T-vector. The result of the sensitivity study showed that 70pg was the lowest concentration of the positive control plasmid during PCR. The specificity results indicated no cross reaction with the genomic DNA of the other bacteria.

Results of this study showed a high specificity and sensitivity of PCR method for the rapid and precise detection of N. meningitidis. This assay as an important supplement would be substituted the previous molecular and time consuming phenotypic assays used in our laboratory and work. in contrast of.

Crimean-Congo hemorrhagic fever (CCHF) is a potentially fatal zeonosis disease caused by a tick-borne virus from the Bunyaviridae family. The CCHF virus is transmitted to humans through the bite of the Ixodid ticks or contact with the blood or tissues of CCHF patients or infected livestock. Human infections begin with the nonspecific febrile symptoms, but progress to a serious hemorrhagic syndrome with a case fatality rate of 2-50%. As Rapid and precise diagnosis approach is critical for control, treatment and detection of patients and is useful for epidemiological studies. We conducted a review study on the different research works on diagnosis field in the world and Iran scientific databases, according to laboratory diagnosis of CCHFin bio safety, sampling and diagnosis assays aspects. Laboratory Diagnosis of CCHF is based on three approaches: Virus Isolation, Serological assays (IIF, RPHA, and ELISA) and antigen detect assays (ELISA, RT-PCR, Real Time RT-PCR and Microarray) Early diagnosis is critical for patient therapy, prevention of potential nosocomial infections and in biological attacks. Also, the ability to distinguish between an infection with a reassortant and a non-reassortant variant may have prognostic value. According to Iranian health center facilities, we should design and established domestic methods in each three approaches especially in BSL4 facilities in Iran.

Salmonella enterica serotype Typhi is the causative pathogen of typhoid fever. Morbidity and mortality rates of the disease are 16×106 and 6×105 cases per year, respectively. Proper diagnosis of the disease and its suitable treatment have critical roles in morbidity and mortality reduction, especially in the developing countries. The traditional methods for detection of the organism are time consuming with low sensitivity. The aim of the present study is to design a new polymerase chain reaction (PCR) method for rapid detection of the organism.

Target specific viaB genes were used for primer designing using Generunner software. PCR tests were sat up using the standard Salmonella typhi genome. Besides, specificity of the method was determined using negative control bacterial genomes. For construction of positive control, determination of sensitivity of the method and limit of the detection, PCR products were cloned in a pTZ57RT plasmid vector.

The agarose gel electrophoresis of PCR products showed 441 bands for the viaB genes. PCR tests for control negative genomes did not create any band on the agarose gel. Results of PCR, enzymatic digestion and sequencing confirmed the cloning process and the positive control construct.

Considering the disadvantages of the classic methods for detection of the organism and the advantages of the molecular methods, the diagnostic kit proposes a suitable tool for rapid detection of the Salmonella typhi.

Yersinia pestis, the causative agent of the zoonotic plague infection, is a major public health concern both as a threat and potential bioweapon. The objective of the present study was to establish a uniplex and multiplex - polymerase chain reaction (PCR) test for the specific detection of Y. pestis. PCR reactions performed by three pair primers which targeted the caf and pla genes located on the pFra and pPst plasmids and the irp chromosomal gene located on the ‘pathogenicity island’. After TA cloning of the PCR products, the test’s limit of detection (LOD) was determined. For evaluating the specificity, PCR reactions were performed with negative control bacteria. Assays were performed with the genome of Y. pestis which produced three DNA fragments of the expected sizes 00, 400 and 50 bp which corresponded to the irp, caf and pla genes, respectively. The lower LoD was 70 copy numbers for the caf gene and for the pla gene. In PCR reactions that used negative control bacteria, detectable fragments were not observed. Our method clearly discriminated Y. pestis DNA. The rapidity, specificity and sensitivity of this procedure suggest that it can serve as a useful alternative method for the inoculation of laboratory animals or the use of specific culture media for routine plaque surveillance and outbreak investigations. Another vital result of this study was the establishment of Y. pestis molecular detection technique in Iran.