Background and Aim: Isolation of genomic DNA from bacterial cells is one of the processes typically performed in most biological laboratories and there are different methods to do it. In this study, two methods including phenol-chloroform, and magnetic nanoparticles, were used to extract genomic DNA of Staphylococcus aureus. Materials and Methods: In the present study, the standard strain of Staphylococcus aureus ATCC 25923 was used for extraction of genomic DNA by phenol-chloroform and magnetic nanoparticles methods. Nanodrop and electrophoresis on agarose gel were used to evaluate the quality and concentration of extracted DNA. Results: The concentration of extracted DNA by phenol-chloroform and magnetic nanoparticle) SiO2/Fe3O4) methods were obtained 550. 4 and 131. 6 µ, g/ml respectively. Conclusion: From the findings of this study it can be concluded that due to the thick wall of Staphylococcus aureus, genomic DNA extraction by magnetic nanoparticles (Fe3O4/SiO2) have acceptable concentration and purity for molecular processes such as PCR, and can be used as an alternative to other extraction methods.

Sarcocyst is one of the most important protozoa belonged to apicomplexa. This parasite is prevalent among warm blooded animals throughout the world. In the present work, sarcocystis gigantea was identified by amplification of 18s rRNA gene using PCR- RFLP. In this regard macroscopic cysts of sarcocystis were collected from esophagus and intra costal muscles of sheep in shahriar slaughterhouse. Then genomic DNA of the parasites was extracted from specimens using phenolchloroform method. 18s rRNA gene was amplified with specific primers. For RFLP two restricted enzymes of MSPI and MOBI were used and the patterns were analyzed accordingly. According to the resulted band, all the specimens were identified as sarcocystis gigantea. This is the first report of molecular identification of sacrocystis gigantea in Iran.

Objective: To evaluate presence of different subtypes and genetic variations of JC virus in different geographical areas is a useful tool for reconstructing of the genetic information and understanding of the evolution of the virus and also in tracing of the last and present history of human immigration. Materials and methods: This study aimed to investigate the reactivation of different genotypes of JC virus in kidney and its excretion in the urine of the 50 pregnant and 50 non-pregnant women. Phenolchloroform method was used to extract DNA. Oligo 7 and MEGA 7 software were used for designing nested PCR specific primers based on vp1 capsid gene, and construction of phylogenetic tree, respectively. Fisher’ s exact test was used for statistical analyses. Results: All of the positive samples were sequenced and according to them, genotypes 1 and 3 of the virus were detected for the first time in pregnant and non-pregnant women in Asia. The frequency of genotypes 1 and 3 were 14. 28% and 85. 71% respectively. Conclusion: For the first time genotype 3 was reported as the frequent genotype in pregnant women in Asia. Confirming these needs more studies particularly with a higher number of cases and full genome sequencing of isolated JCVs.

Contagious Caprine Pleuropneumonia (CCPP) is one of the common infections in the middle east regions. So far, there has not been received any report about isolation and identification of these agents in Iran. The aim of this study is to diagnose and isolate mycoplasma agents in suspected goat flocks. Total of 100 pneumonic lung specimen from 20 CCPPsuspected flocks were collected from abbatoirs close to Kermanshah during 1384-1386 and had been sent to Microbiology Lab. Gross lesions showed hepatization with grey and white lesions (consolidation) and motley appearance with or without fibrin. The minced tissue were inoculated to PPLO broth agar. After multiple passages, typical mycoplasma colony was isolated from 4 flocks (22/2%). Mycoplasma DNA was also extracted based on phenolchloroform method and subjected to generic PCR with specific primers. In addition to the perivious positive samples from tissue culture, 5other flocks also showed contamination with Mycoplasma organisms in PCR tests(45%). Then, the samples were determined for Mycoplasma mycoides cluster infection, M. capricolum capripneumonia and M. mycoides mycoides (L.C), using M. agalactia as negative control, with specific primers in PCR, there has showed no contamination to these strains. However, to declare "free status " from CCPP in goat flocks requires more developed researches and much more samples in further investigation.

Background: Chronic hepatitis B virus (HBV) infection is a multi-factorial disease that is accompanied with serious clinical complications. Host’s genetic background, especially immune– genetic factors, is critical in the pathogenesis of infection. Gamma interferon ((INF-g) and its receptor have an important role in immune response to the virus and clinical course of the disease. The aim of this study is to investigate the association between single nucleotide polymorphism -611G/A located in promoter of gamma interferon receptor1 gene (INFGR1) and chronic HBV infection.Materials and Methods: In this Case Control study, genomic DNA from peripheral blood samples of 150 chronically HBV infected patients and 150 healthy controls was extracted by phenolchloroform method. DNA analysis was performed by PCR-RFLP method and P<0.05 was considered significant.Results: After stages of genotyping and statistical analysis, a significant difference was observed between patient and control group, so that genotype GG was higher in the control group compared to the patient group.Conclusion: The host’s immune-genetic background can play an important role in the pathogenesis of infectious disease. Variations in INFGR1 were related to several diseases. The results showed that the presence of GG allele is accompanied by a decrease in susceptibility to chronic HBV infection.

selection method using DNA markers is an assured method in breeding process via preparation of linkage map and QTLs determination of effective genes on quantitative traits control. This method can be used in the silkworm, since it has economical-productive and polygenic traits. It was used in this research for determination of QTLs of shell cocoon percentage by AFLP markers, 20 compounds of two primers (TaqI & PstI) that selected from 81 compounds in 33, 36 and 34 of F2 individuals of three mulberry silkworm populations which these progency was produced by mating of two Lemon Khorasan (as female) and 107 (as male) lines as pure parents. The extraction of DNA was done using phenolchloroform and extracted DNA was digested by TaqI and PstI restriction enzymes, so they linked to suitable adaptors and selected propagation of all samples of DNA was done by means of all of the primer compounds. The propagated samples were transferred to the acrylamid gel 6% and the linkage maps of studied populations were drawn based on matrix formation (number of progency of F2 population× number of appeared poly-morphic bands) by Map Manager and Cartographer ver.2.5 Softwares after genotyping of individuals and ultimately is identified number of possible locations of studied trait controller based on QTL analysis through Composite Interval Mapping in Likelihood Ratio Test (LRT) >13 and detected 4, 2 and 5 QTLs respectively within recessive-overdominance limitation.

Bream (Abramis brama orientalis) is one of the species of Cyprinidae that lives in Caspian Sea and its watershed. During the recent decades by several reasons such as over fishing, pollution increase, destroying of spawing regions, the fish stock decreased in south coast of Iran, Caspian Sea. Because of that, during the previous years, Iran Fishing Organization decided to recover the fish stock by the usage of artificial reproduction of a broodstocks of Bream population in Iranian Caspian Sea coast. So it hersbeen decided that by entering Bream from Azerbaijan Republic coast an increase can occur in population diversity of that fish, thereby from each 3 mentioned regions 30 were hunted and carried to the genetic laboratory. After extracting DNA by Phenolchloroform method for genetic-molecular research PCR-RFLP technique was use on a mitochondrion genome piece by the length of 3500 contains tRNA-glu, tRNA-leu, ND 5/6 and Cyt b. Between 17 used enzyme, 4 enzyme DraI, BCLI, Hae III and BanII showed diversity which generally 6 haploids was recognized. The most nucleotide diversity 0.58% by presence of all haploids was observed in Azerbaijan Republic coast samples while Anzali wetland and Iranian Caspian Sea coast, Bream had no diversity. Also, statistical analysis by the usage of Monte Carlo test by 10000 repetition showed a significant differences between Azerbaijan Republic coast fishes (p<0.0001) but there was no significant differences between two regions (p>0.5).

Background: To identify the fasciolid species by morphometric and molecular methods in Zanjan, northwest of Iran.Methods: Adult Fasciola worms (n=584) were obtained from cattle and sheep in Zanjan slaughterhouse in 2007. Living flukes were washed, then worms' images were taken by 3CCD camera and finally apical zone of each worm was obtained. Morphometric values such as body length, body width, body area, body perimeter and the distance between ventral sucker and posterior end of body were obtained using Auto- CAD image analysis software. Total gDNA was extracted from individual flukes by modified phenolchloroform method. PCR amplification of ITS2 fragment was performed the isolated DNA samples and the amplicons were consequently subjected to RFLP assay and nucleotide sequencing to distinguish between fasciolid species.Results: Mean of morphometric values in flukes from sheep was greater than those of cattle. Accordingly, the identified species included 31% F. hepatica-like, 7% F. gigantica-like and 62% intermediate forms. However, ITS2 fragment of 535 amplified specimens, showed no variation at the species-specific nucleotide sites 230, 340 and 341. The amplified fragment composed of partial 5.8S sequence (62bp), the complete ITS2 sequence (361bp) and partial 28S sequence (34bp). The nucleotide contents of ITS2 region were 69 A, 116 T, 81 C and 95 G and the average G+C content was approximately 49%. Comparing of ITS2 sequences with the BLAST GenBank database, also confirmed that all specimens were F. hepatica. Conclusion: A simple and rapid PCR-RFLP assay can be used for distinguishing between these species.

Objective: This study was carried out for identification of Eimeria spp isolated from poultry breeder farms in Iran by PCR. Design: Pop-Gene Analysis. Animals: Poultry breeder farms. Procedures: A total of 114 litter samples from poultry breeder farms without previous exposure to anticoccidial vaccine were collected randomly from relatively five different climate regions of Iran. DNA was extracted from oocysts of samples, using phenolchloroform and proteinase-K. Four pairs of specific primers, designated from Internal Transcribed Spacer-1 (ITS I) regions of ribosomal DNA of E. acervulina, E. brunetti, E. necatrix, and E. tenella and one pairs of universal primer BSEF-BSER which amplify ITS 1 of different Eimeria were used in PCR assay. In tests on purified genomic DNA from all species of Eimeria isolated from infected samples, each of four primer pairs amplified the ITS 1 region of their respective target species only. The PCR products were analyzed by agarose gel electrophoresis. Results: DNA fragments in sizes of 320 pairs (E. acervulina), 311 pairs (E. brunette), 384 pairs (E. necatrix) and 287 pairs (E. tenella) were detected on agarose gel electrophoresis. Universal primer pairs also amplified ITS 1 of five Eimeria which isolated from infected samples. Laboratory implicatious: The results of this study were showed that PCR technique is a conventional method, faster, technically easier and very cheaper than other methods to identify the Eimeria spp. Finally, this technique can be recommended to be a routine work in well equipped veterinary diagnostic labs in IRAN.

Introduction: Annually, more than 14 million people are reported to be infected with Leishmaniasis all over the world. In Iran, this disease is seen in the form of cutaneous and visceral leishmaniasis, of which the cutaneous form is more wide spread. In recent years, cutaneous leishmaniaisis is diagnosed by PCR utilizing specific primers in order to amplify different parasite genes including ribosomal RNA genes, kinetoplast DNA or tandem repeating sequences. The aim of this research was to detect early stage cutaneous leishmaniasis using Multiplex-PCR technique. Methods: In this study, 67 samples were prepared from patients with cutaneous leishmaniasis. DNA was extracted with phenolchloroform. Each specimen was analyzed using two different pairs of PCR primers. The sensitivity of each PCR was optimized on pure Leishmania DNA prior to use for diagnosis. Two standard parasites L. major and L. tropica were used as positive control. Results: DNA amplification fragments were two 115 bp and 683 bp for AB and UL primers, respectively. The sensitivity of two primers was not equal for detection of L. major and L. tropica. The sensivity of PCR with AB primer was 35 cells, while that for UL primer was 40 cells. Conclusion: The results of this study indicate that PCR is a sensitive diagnostic assay for cutaneous leishmaniasis and could be employed as the new standard for routine diagnosis when species identification is not required. However, the ability to identify species is especially important in prognosis of the disease and in deciding appropriate therapy, especially in regions where more than one type of species and disease are seen by clinicians.