Tarkhineh is one of the traditional foods in Iran and a rich source of probiotic bacteria. The objective of this study was to identify the probiotic bacteria isolated from Tarkhineh using 16S rRNA gene sequencing and molecular typing with repetitive extragenic palindromic–PCR (REP-PCR). In total, 20 different bacteria were isolated from traditional dairy products and Tarkhineh. Molecular identification of the isolates was carried out by 16S rRNA gene sequencing, and DNA sequences of isolates deposited in GenBank. The REP-PCR reaction by REP1R-I, REP2-I and REP1R-I+REP2-I markers was performed for fingerprinting and characterization of the isolates. Unweighted pair group method with arithmetic mean (UPGMA) clustering methods were performed based on Dice similarity. The REP1R-I primer grouped isolates into three, and REP2-I and REP1R-I+REP2-I grouped all isolates into four main clusters in dendrograms. In all analyses, isolates of Lactobacillus casei, Lactobacillus brevis, Lactobacillus plantarum, and Entrococcus facium formed separate clusters. The results of sequencing corresponded to clustering in the dendrogram. According to the results, REPPCR is an accurate technique for determining the genetic diversity of lactic acid bacteria species.

In the present study, Randomly Amplified Polymorphic DNA (RAPD) and Repetitive Extragenic Palindromic sequence-based Polymerase Chain Reaction (REPPCR) were used to characterize 131 isolates of Pasteurella multocida, originating from different healthy and diseased animal species obtained from several geographical regions of Iran. The RAPD and REP-PCR generated amplified products in the range of 300 to 3400 bp and 200 to 2850 bp, respectively. Among all of the P. multocida isolates, cluster analysis revealed that 63 clusters and nine untypable isolates and 81 clusters and six untypable isolates were produced with RAPD and REP-PCR methods, respectively. The results indicated that the REP-PCR method showed a slightly higher level of discrimination power in differentiating of P. multocida isolates as compared with RAPD. The results showed that a considerable level of genetic diversity exists among P. multocida isolates even in the isolates with the same animal or geographical origins. There was no host- and region-specific pattern. In addition, the isolates obtained from the healthy and diseased animal did not reveal any correlation genotypic profiles, which could be supported by the hypothesis that P. multocida is a strictly opportunistic pathogen. In conclusion, because of a large amount of genetic heterogeneity in the P. multocida isolates, Pasteurellosis may be caused by different clones in the same herd or animal.

Background and Aim: DNA extraction is the first step in the genetic engineering research and obtains a suitable protocol for the preparation of a purified DNA which is essential. In this context, many manual and kit methods were provided. The aim of this study is to achieve a faster and low cost method for DNA extraction of Gram-positive bacteria and gram-negative bacteria. We compared four DNA extraction methods, Kit extraction, phenol chloroform, using detergent laundry brand tag and boiling.Materials and Methods: In this study, bacterial suspensions of Staphylococcus aureus and Vibrio cholerae were produced similar to McFarland 0.5 turbidity. Bacterial DNA was extracted using four different methods and three times for each. Using spectrophotometer and gel electrophoresis were measured concentrations and quality of DNA extraction product accordingly. In order to evaluate the efficiency of DNA extraction method, PCR, ERIC PCR and REP-PCR were performed on some of housekeeping genes downstream regions.Results: The concentration of the extracted DNA and results of PCR, ERIC-PCR and REPPCR were different between gram-positive and gram-negative bacteria significantly (P<0.05). ERIC PCR and REP-PCR efficiency of the boiling and chloroform extraction methods was disrupted only in ERIC PCR. No deficiency was observed in Sinagen kit method.Conclusions: The findings of this study show that despite the availability and lower cost of manual methods, these methods can be substitute for kit method.