Background & Objective: Knowledge of allele groups and specific alleles present in individuals has important implications in organ and stem cell transplantation and in disease association studies. In organ transplantation application of molecular HLA typing allowed to improve typing quality, leading to a more precise matching assessment with better clinical outcomes. In this study, we have developed the REVERSE DOT-BLOT (RDB) hybridization technique as a rapid and simple method for detection of HLA-DRB1*01 group alleles.Materials & Methods: For high-resolution typing of HLA-DRB1*01 group alleles, we performed amplification of the alleles of this group using allele-specific primers. Productive DNA amplification occurs if an allele perfectly be complementary to the two primers chosen is present. This technique is often referred to as PCR-SSP. Then for specific discrimination between these alleles (HLA-DRB1*0101, 0102, 0103, 0104), we hybridized PCR product, labeled with biotinylated primers during the amplification, with spots of immobilized probes on a membrane. Variety of immobilization methods could be used. We developed a covalent attachment between 5’-amino probes and carboxylated membrane surfaces. The presence of the PCR product bound to the specific probes at specific locations is detected using streptavidin-AP conjugate and NBT/BCIP as a chromogenic substrate.Results: The presence of each specific allele was detected by the appearance of a DOT on the membrane. Obtained results were compatible with those of expected for standard allele samples. Furthermore, results obtained for several other samples which were typed by our technique, were confirmed using sequencing.Conclusion: Our results suggest that HLA typing by RDB method has proved to be a high-resolution, high specificity, rapid and accurate, suitable for clinical application with a greater precision than serological techniques.

As a rule the reference genes used in gene expression analysis have been selected for their housekeeping roles, but the variation observed in most of them is a major obstacle to their effective use. It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. In this study, suitability of seven wheat housekeeping genes for normalization of mRNA expression in wheat leaves infected by Mycosphaerella graminicolawas investigated. Expression level of Actin, Rubisco, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Translation Elongation Factor 1α (TEF- 1a), a-Tubollin, eukaryotic release factors 1 and 3 (ERF 1 and ERF 3) genes were examined by REVERSE northern DOT BLOT method. Expression stabilities of the reference genes were statistically analyzed by Ecxel and SAS softwares. a-Tubolin, TEF- 1a and Actin were the three most stable genes whereas the expression of Rubisco and GAPDH had the least stability. The presented comprehensive data on changes in expression of various wheat housekeeping genes in wheat-M. graminicola phatosystem facilitate selection of reference genes for REVERSE northern DOT BLOT method.

Background and Objective: Alpha thalassemia is one of the most common hemoglobin disorders. Some combination of alpha globin gene mutations may cause HbH disease with severe anemia or intermediate thalassemia. genotype common deletions are routinely tested for suspicious alpha thalassemia couples but because of lack of information about the nature and frequency of point mutations and higher expenosor of sequencing, less attention was paid to them. This study was done to determine the prevalence of common point mutations of alpha globin gene in Babol, Iran.Materials and Methods: This descriptive study was carried out on DNA of 153 adult suspected to a-thalasemia with deleted a-golobolin gene referred to genetic laboratory in Babol, Iran during 2005-09. a1 and a2 genes were amplified by using specific biotinilated primers by PCR method. PCR products were assayed using 11 specific probs corresponding to common point mutations in alpha gene (C19, IVSI (-5nt), C59, Hb constant spring, Hb Icaria, Hb seal Rock, IVSI (148), C14, poly A (-2bp), poly A2, Poly A1) and fixed on byodine C membrabe. Hybridization between the probes and PCR products was visualized after a colorimetric reaction using of conjugated streptavidin peroxidase and TMB (tetra methyle Benzidine) and H2O2.Results: The prevalence of point mutations in poly A2, 5nt, Hb constant spring and poly A1 were 28.75%, 14.38%, 7.84% and 2.61%, respectively.Conclusion: Point mutation in alpha globin genes was detected in %53.60 out of 153 adults suspected with alpha thalassemia without common deletion mutations.

Background: Human leukocyte antigen (HLA) class II molecules are encoded by genes in the human major histo compatibility complex (MHC) which is located on the short arm of chromosome 6. The three major class II loci DR, DQ and DP consist of separate A and B genes encoding ab heterodimers responsible for the presentation of peptide antigens to the T cell receptor (TCR) repertoire. The HLA class II molecules have a well-defined role in the cellular immune response. The HLA class II genes are highly polymorphic and most of the polymorphisms are localized in the second exon . The HLA class II molecules have a well-defined role in the cellular immune response and in the bone marrow and organ transplantation.Methods and Materials: To evaluate the accuracy and resolution of different molecular HLA typing methods; 7 WHO DNA samples with known HLA-DRB genotype were typed for DRB genes with PCR-SSP (Sequence specific primers), PCR-SSP-RFLP and PCR-RDB (REVERSE DOT-BLOT) techniques.Results: The result showed that PCR-SSP and PCR-SSP-RFLP methods were not able to identify all the alleles of the WHO DNA samples while PCR-RDB technique detected all the alleles of the WHO DNA samples with great precision.Conclusion:These results clearly indicate that gnomic typing of HLA class II by PCR-RDB is very sensitive and accurate technique in HLA typing for bone marrow and organ transplantation.

Background: Diagnosis of cryptosporidiosis is based on the use of the routine microscopic methods, but these methods possess poor sensitivity and also need an expert technician. The aim of the present study was to determine sensitivity and specificity of DOT BLOT method for diagnosis of bovine cryptosporidiosis.Methods: In this study 131 fecal samples were collected from suckling calves during summer 2014. The collected samples were concentrated using formalin ether method. The samples were then examined by modified-Ziehl-Neelsen (mZN) and finally all samples were examined by DOT BLOT methods.Findings: Oocysts of Cryptosporidium spp. were found in 40 (30.5%) out of 131 fecal samples using mZN staining method but 49 (37.4%) of the samples were positive for Cryptosporidium antigen using DOT BLOT method.No significant differences were observed among the results of the two methods and the sensitivity and specificity of DOT BLOT calculated as 87% and 86%, respectively.Conclusion: Based on the results of the present study, DOT BLOT method showed rather equal performance as modified-Ziehl-Neelsen method.

Background & aim: Human papillomavirus is the most common sexually transmitted disease and the main risk factor for malignant and malignant lesions in the cervix. The aim of the present study was to determine and evaluate the prevalence of human papillomavirus in women with abnormal pap smear referred to Shahid Mofatteh specialized clinic by Multiplex PCR & REVERSE DOT BLOT Hybridization in Yasuj, Iran. Methods: The present descriptive-analytical study was conducted during the years 2015-2016. A total of 67 abnormal cervical cytology specimens including 51 ASC-US specimens, 9 LSIL specimens and 7 HSIL specimens were collected and analyzed by Multiplex PCR & REVERSE DOT BLOT Hybridization. The collected data were analyzed using chi-square test Result: The overall prevalence of human papillomavirus (HPV) was 61. 2%. 36 (87. 8%) women were infected with high-risk types of human papillomavirus, 26 (63. 4%) with low-risk types and 2 (4. 9%) with unknown types. The most common high-risk types were HPV 39 (43. 9%), HPV66 (31. 7%), HPV31 (26. 8%) and HPV18 (14. 6%), respectively. HPV16 was detected in only 2 samples (4. 9%). Conclusion: The results of the present study indicated a high prevalence of papillomavirus infection in women with abnormal Pap smear. In addition, different high-risk genotypes of this virus were identified compared to other studies in Iran. Further samples from wider geographical areas are needed to study the molecular epidemiology and genotypes of human papillomavirus.

Objective: Cystic fibrosis and its distribution vary widely in different countries and/or ethnic groups. Common cystic fibrosis transmembrane conductance regulator (CFTR) mutations were reported from Iran, but the northern population was not or underrepresented in those studies. The aim of this study was to determine the frequency of common CFTR mutations in children from northern Iran.Methods: Thirty unrelated Iranian cystic fibrosis patients aged less than 11 years and living in Mazandaran province (in Iran) were screened for 5 common CFTR gene mutations. deltaF508, N1303K, G542X, R347H and W1282X using REVERSE DOT BLOT method.Findings: Only one mutation, DeltaF508, was found in 7 patients accounting for 21.7% (13/60) of alleles.Conclusion: These findings can be used for planning future screening and appropriate genetic counseling programs in Iranian CF families.

Background: Human papilloma virus (HPV) is the most common sexually transmitted viral infection worldwide. On the basis of HPV clinical associations, this disease is divided to 2 types; high risk and low risk. high risk HPV (HR HPV) is the causative agent of cervical intraepithelial Neoplasia (CIN) and cervical cancer. low risk HPV (LR HPV) is the causative agent of diseases, such as anogenital warts and oral papillomas. Polymerase chain reaction (PCR) is one of the molecular diagnostic methods for detection of HPV DNA that has many advantages. Objectives: This study aimed at exploring the frequency of HPV genotypes using polymerase chain reaction REVERSE DOT BLOT (PCR RDB) method on cervix and wart samples obtained from patients in Isfahan, Iran. Methods: The population included 111 females and 7 males. These samples were collected during 2011 and 2013. DNA was extracted from these samples and PCR was performed using REVERSE DOT BLOT (RDB) method to detect HPV types. Results: With this molecular diagnostic method, different HPV types were detected in 45 samples out of 118. In 18 samples, coinfection with different HPV types was detected. Data analysis exhibited a significant correlation between age and HR HPV infection (P value < 0. 05). However, no significant relationship was detected between LR HPV and age (P value > 0. 05). In this study, among all HPV types, HPV 16 was detected more frequently than the other HPV types. Conclusions: The PCR-RDB method has the ability to diagnose HPV infection by screening Pap smear samples and to determine the risk factor of detected HPV genotypes. This study showed that HPV16 was 1 of the most common agents in Iran, similar to other countries.