Expression of mRNA-TLR-5 Gene in Patients with Endometriosis using Real-time PCR in Tehran, Iran.

BACKGROUND: Endometriosis is one of the common diseases of women, especially in reproductive age, and it is one of the most important causes of infertility in women. The aim of this study was to investigate the level of mRNA-TLR-5 expression in women with endometriosis. METHODS: The present study was performed in Nikan Hospital, Tehran, Iran, in 2021. The samples of endometrial mucosa for the eutopic group and an ovarian endometriotic cyst for the ectopic group were obtained from the patients who underwent laparoscopic surgery at the Fetal Infertility Center and were diagnosed with endometriosis. Normal endometrial samples were also obtained from patients who had no history of infertility and underwent laparoscopic TL surgery for reasons other than endometriosis such as ovarian cysts (control group). After RNA extraction and cDNA synthesis, TLR-5 gene expression was evaluated by the Real-Time PCR method. RESULTS: Based on the results of the comparison of TLR-5 gene expression in all three ectopic, eutopic endometrium, and control groups by Real-Time PCR, it was found that the TLR-5 gene expression is significantly higher in ectopic samples than in the other two groups, but there is a significant difference between two utopic and control groups. CONCLUSION: The increase in TLR-5 expression in the ectopic group can probably be a reason for reducing the apoptosis of cells entered into the peritoneal cavity and creating an environment for the survival and proliferation of these cells.

Molecular design real time loop-mediated isothermal amplification method for rapid detection of Neisseria meningitidis.

INTRODUCTION: Detection of Neisseria meningitides by using conventional methods is time consuming and laborious. Development of a realiable and rapid method for prompt control and prevention of meningococcal disease is required. Although PCR and real time PCR methods have been developed; they require electrophoresis or expensive Devices. LAMP is a simple gene amplification method which can be performed at a single temperature without the need for thermal cycling. OBJECTIVE: We aimed to develop a quantitative real-time LAMP assay for detection of N. meningitides and accurate quantification of the bacterial load in patients with meningococcal disease. MATERIAL AND METHODS: the LAMP reaction was set up and optimized by four primers were designed. Amplification results were assessed by obtained real time turbidity graphs from each LAMP reaction tube using real time turbidimeter apparatus. Standard curve was generated from turbidity graphs corresponding to ten-fold serial dilution of crgA gene containing recombinant plasmid. RESULTS: by LAMP assay just N. meningitides isolated, whereas no amplification was obtained with negative control isolates, and this indicating 100% specificity. The limit of detection (LOD) of our LAMP assay was found to be ~ 5 copies of crgA gene per reaction.  REAL LAMP Analysis of the standard curve revealed excellent linear correlation between gene copy number and time threshold. With a coefficient correlation equal to 0.92.

Detection of Arctic and European cluster of canine distemper virus in north and center of Iran.

Canine distemper virus (CDV) creates a very contagious viral multi-systemic canine distemper (CD) disease that affects most species of Carnivora order. The virus is genetically heterogeneous, particularly in section of the hemagglutinin (H) gene. Sequence analysis of the H gene can be useful to investigate distinction of various lineages related to geographical distribution and CDV molecular epidemiology. Since vaccination program is conducted only in large cities of Iran, CD still remains as one of the major causes of death in dogs in this country. In order to monitor H gene, CDV has been detected in 14 out of 19 sampled dogs through the amplification of nucleoprotein (NP) gene in nested-PCR assay. In the next step 665 bp of H gene was amplified in 9 out of 14 NP-gene positive dogs. Phylogenetic analysis distinguished two distinct CDV genotypes in Iran. JN941238 has been embedded in European cluster and JN941239 has been embedded in Arctic cluster. Nucleic analysis has been shown high difference among both Iranian CDV lineages with CDV vaccine strains.

Development and Application of an In-house Line Probe Assay for Hepatitis C Virus Genotyping.

BACKGROUND: Hepatitis C virus (HCV) is the major cause of chronic liver disease. HCV is a single stranded positive sense RNA of approximately 9.6 Kb. Because of high conservativeness of 5΄untranslated region of HCV genome, it is widely used for virus genotyping. Different methods are used for the virus genotyping, but all involve some difficulties. OBJECTIVES: The aim of the present study was to develop an in-house reverse hybridization method as a line probe assay, for HCV genotyping. MATERIALS AND METHODS: Sixty serum samples were collected with newly diagnosis of HCV infection. Genotyping process had already been performed for the samples using RT-PCR RFLP method. After total RNA extraction from the samples and cDNA synthesis, nested PCR method was applied for amplification of the target sequence on the 5΄UTR. In the nested PCR, biotinylated oligonucleotides were used as inner primers. Optimized concentrations of the biotinylated inner primers (as positive control), two universal and seven specific probes were spotted onto nylon membrane stripes in a defined pattern. Hybridization process was conducted between the probes and the denaturized biotin labeled PCR products. Finally, the stripes were developed by using streptavidin conjugated alkaline phosphate as a signal generating agent. To determine the diagnostic sensitivity and specificity of the home made LiPA, a panel containing 60 confirmed sera with positive results for HCV (and PCR-RFLP genotyped) was subjected to evaluate. RESULTS: Agarose gel electrophoresis of the nested PCR products using the outer and inner primers showed 305 and 234 bp fragments respectively. After performing hybridization and detection processes on the prepared strips, the colored bands were formed for the positive control, universal probes and the corresponding genotypes. HCV genotype results were found to be in 100% concordance through studying 60 sera that were successfully typed by the two methods. P-value of 0.045 conveys that the two methods were the same and had no significant difference. CONCLUSIONS: The most common genotyping method in Iran is RT-PCR RFLP. Given the results and advantages of this homemade technique, such as high specificity and sensitivity, ability for detection of most genotypes, it provides possibility of evaluating much of the isolates without needing electrophoresis stage.

Design of an improved multiplex PCR method for diagnosis of enterohaemoraghic E.coli and enteropathogic E.coli pathotypes.

AIM: We aimed to develop a multiplex PCR assay for specific detection of EPEC and EHEC pathotypes based on specific marker genes. BACKGROUND: About 2.5 million infant's morbidity in developing countries occurs by E.coli pathotypes because of diarrhea and intestinal diseases. The traditional phenotypic methods are time consuming and sometimes detection and differentiation of the pathotypes are not done easily. Multiplex PCR technology is used as a sensitive, specific and rapid molecular method for detection of various pathogens. PATIENTS AND METHODS: PCR reactions were performed with primers which targeted the virulence genes selected for each category (stx 1 , stx 2 genes for EHEC and bfpA for EPEC). For preparation of a positive control, the PCR products were cloned in pTZ57R/T plasmid. The same PCR reactions were done but in presence of genomes of various negative control bacteria for evaluation of test specificity. RESULTS: As expected, gel agarose electrophoresis of PCR products of the stx 1 , stx 2 and bfpA, showed 329bp, 586bp and459bp bands respectively. Result of amplification using negative control genomes as template was negative. CONCLUSION: The multiplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the four main pathotypes of E. coli. This assay will replace the previous molecular genetics methods used in our laboratory and work as an important supplement to the more time consuming phenotypic assays.