Iranian Journal of Biotechnology
Year:2013 | Volume:11 | Issue:2 (42)|Papers Count:10

For many years, assessment of olive oil quality and its authenticity confirmation has been of great importance to guarantee olive oil safety in compliance with legislations. These include the investigation of olive oil purity and quality by analyzing several parameters such as fatty acid composition, sterols, phenolic compounds, triacylglycerol and etc, through performing conventional and advanced hyphenated techniques.

Background: Due to some limitations of serological methods in diagnosis of patients infected with HIV-1 (human immunodeficiency virus) and HCV (hepatitis C virus), it is profoundly important to use molecular methods for the detecting of these infectious agents. However, the most significant problems are the exorbitant cost of these methods and the need of a thermocycler which is an expensive instrument.Objectives: The current research recruits a multiplex nucleic acid sequence base amplification (NASBA) in order to simultaneously detect HIV-1 and HCV genomes in patients’ plasma samples. Sensitivity and specificity of this method have been evaluated using clinical samples.Materials and Methods: A multiplex NASBA assay for simultaneous detection of HCV and HIV-1 by the use of specific primers were designed and validated. A well-conserved region in the HIV-1 pol gene and 5’-NCR of HCV genome were used. A total of 40 samples of HIV-1 (20 samples) and HCV (20 samples) were used in the NASBA assay. The specificity and sensitivity of the assay were evaluated. Results: Our results have demonstrated that the primers used in the assay had no interrelation with each other and other possible interfering agents in the assay. The analytical sensitivity of the assay for both HIV-1 and HCV was determined to be 1000 copies/mL and the clinical sensitivity and specificity were 93.3% and 100%, respectively.Conclusions: By exploiting this multiplex NASBA assay, it is possible to detect HIV-1 and HCV infection/co-infection in patients’ plasma with a suitable sensitivity and specificity. Furthermore, due to its simplicity and multiplexing feature, it could be used in limited access laboratories in a cost-effective manner.

Background: Brucellosis is still considered as one of the major zoonosis afflicting Syrian health and economy. This disease is caused by members of the genus Brucella which are gram-negative bacteria living facultatively within mammalian cells during infection. Objectives: In this paper, a strategy was developed to introduce a new generation of binders called Nanobodies (Nbs) in our combat against Brucella. Nbs are genetically engineered camelid-derived single-domain antibody fragments that are very stable and highly soluble, making them promising tools in numerous biotechnological and medical applications. Materials and Methods: In our previous work, three Nb-displaying phages (Nb-phage), referred to as NbBruc01, 02 and 03, were retrieved by a phage display panning of a Nb library constructed from Brucella- immunized camel. In this work, soluble Nbs were produced after recloning their genes in protein expression plasmid followed by purification with affinity chromatography. Results: Interestingly, two of these soluble Nbs (NbBruc02 and 03) were able to detect Brucella antigens from two main Brucella species (B. abortus and B. melitensis) and distinguish them from those of Yersinia. This is remarkable as the camel IgG failed in such antigen discriminations. High similarity, mainly in the structure of lipopolysaccharides (LPS) of these different types of bacteria, causes regular serum cross reactivity and thus lack of specificity urging the need for more accurate diagnostic techniques, e.g. a multiplex PCR. Furthermore, NbBruc02 and 03 targets may represent Brucella immunodominant proteins as shown by immunoblotting. Conclusions: In addition to their own importance, identifying these antigenic targets will open new perspectives for diagnosis, vaccines and treatment of Brucellosis.

Background: Sugarcane is a monocotyledonous crop that is cultivated in the tropical and subtropical regions of the world. One of the most important criteria, influencing the efficiency of the sugarcane transformation is known to be related to physical and biological factors during the transformation procedure. Objectives: The objective of this research was to study the response of callus induction and embryogenic callus production and to identify the major parameters controlling DNA delivery by particle bombardment into sugarcane (Saccharum officinarum L.) cv. NCo310. Materials and Methods: For callus induction and embryogenic callus production, leaf base segments were subjected to in vitro culture medium supplemented with two plant growth regulators (2,4-D and Dicamba). Results showed that 1 mg.L -1 2, 4-D was significantly influential in callus induction and embryogenic callus production. Considering both physical and biological factors, the efficiency of DNA (uidA gene) delivery was assessed by scoring transient GUS (gene (b-glucuronidase)) expression in bombarded tissues. Results: The highest transient GUS expression was obtained when callus was bombarded with the construct harboring rice Act1 promoter, and having 9 cm target distance, 25 inHg vacuum pressure, 1 mm gold particles, 12.5 mg DNA per bombardment and one day pre-culture prior to the bombardment. Conclusions: A bombardment procedure suitable for elite sugarcane varieties was developed, which allowed high-efficiency DNA delivery combined with reduced damage to target tissues.

Background: Taxol is one of the most important anti-cancer drugs, which is obtained from yew trees (Taxus sp.). T+he first step in side chain assembly of taxol is catalyzed by phenylalanine aminomutase, which converts α-phenylalanine to b-phenylalanine. Objectives: In this study, for the first time, we report on the cloning, preliminary expression and characterization of a full-length gene and cDNA encoding phenylalanine aminomutase from Taxus baccata L.Materials and Methods: Comparison of the full-length gene with other ones identified from the Taxus species showed high similarity, particularly with Taxus x media.Results: The results showed that the expression level of this gene in Taxus baccata is very low and therefore this enzymatic step could be a rate limiting step in the taxol biosynthesis pathway. Successful amplification of the cDNA was only obtained from RNA samples isolated from methyl jasmonate elicited suspension cells of Taxus baccata. The cloned cDNA contained a 2064 bp open reading frame encoding a protein composed of 687 amino acids. Sequence comparison analysis revealed that the gene is very similar (98 - 99 %) with respect to the nucleotide and amino acid sequences in different Taxus species and also share the signature active site motif (175ASG177).Conclusions: The predicted structure of TbPAM was analyzed using bioinformatic tools. The results indicated that the protein has similar overall folding to tyrosine aminomutase.

Background: The High Resolution Melting (HRM) method is a new scanning method for detecting unknown changes in DNA and its advantages have persuaded researchers to recruit it as a screening method. Objectives: Here, we developed a HRM method to screen R188H SNP (rs3218536) of XRCC2 and compared the results with a well known PCRRFLP technique.Materials and Methods: Genomic DNA samples from 350 healthy individuals were obtained. PCR-HRM analysis and PCR-RFLP method were performed simultaneously.Results: Three different melting profiles corresponding to three different genotypes recognized by HRM analysis. The results of PCR-RFLP showed no discrepancy. Conclusions: We concluded that the HRM technique can be used as a screening method for rapid discrimination of R188H genotypes in XRCC2 gene.

Background: Cucumber mosaic virus (CMV) from the Bromoviridae family, is one of the most widespread plant viruses in the world. Objectives: In the present study tomato fields in Guilan, Isfahan, Khorasan Razavi, Khuzestan and Tehran provinces were surveyed to determine the presence of CMV subgroups during 2011-2012. Materials and Methods: Out of 305 symptomatic leaf samples tested by Enzyme-linked immuno sorbent assay (ELISA), 147 samples (48.2 %) were found to be infected by CMV with the highest percentage in Khorasan Razavi (67.4%) followed by Khuzestan (50.6%), Tehran (48%), Isfahan (38.2%) and Guilan (34.3%). The coat protein (CP) gene in the 19 sequenced CMV isolates composed of 657 nucleotides (nt) in a size that encodes 218 amino acids. Phylogenetic analysis based on the nt CP gene showed that the ToKz1, ToKz2, ToKz3 and ToKz4 from Khuzestan fell into subgroup IB and the rest of the Iranian isolates including those sequenced in this study fell into subgroup IA.Results: Subsequent analyses showed that the Iranian CMV isolates belonging to subgroup IA of CMV were most related phylogenetically to each other and they were distinct from the subgroup IB and subgroup II isolates. Bioassay on Nicotiana glutinosa and Solanum lycopersicum showed that the symptoms caused by subgroup IB isolates from Khuzestan were milder than those caused by CMV isolates from subgroup IA under this study.Conclusions: In Iran only subgroups IA and II have been reported, however for the first time this study shows the occurrence and phylogenetic relationships of CMV subgroup IB isolated from tomato fields in West Asia, Iran.

Background: Potyviruses are accounted for 40% of viral diseases of various crops including vegetables and legumes. Potyviruses can be transmitted through plant sap, seeds and many aphid species. Due to ease of spread of these viruses detection of such viruses are crucial as to the control of the incited diseases.Objectives: This study compared the efficiencies of two couples of primers in their detection of potyviruses infecting vegetables in North-West of Iran.Materials and Methods: To identify potyviruses infecting vegetables, total RNA preparations from leaves of diseased plants were subjected to reverse transcription (RT) with oligo d (T) 15, M4T or a potyvirus-specific primer, followed by polymerase chain reactions (PCR) with two pairs of degenerate primers including Sprimer/M4 or NIb2F/NIb3R that would yield  ~1700 or 350 bp fragments, respectively. Amplification was achieved from 7 samples when Sprimer and M4 were used, but non- specific fragments were also amplified. However, specific amplifications from 31 samples were achieved with NIb2F2 and NIb3R. PCR products resulting from the use of NIb2F/NIb3R were subjected for cloning and sequencing.Results: BLAST analysis of the sequenced data revealed that the PCR-amplified fragments belonged to Bean common mosaic virus (green bean), Bean yellow mosaic virus (broad bean), Potato virus Y (potato), Watermelon mosaic virus (squash, muskmelon or melon), Zucchini yellow mosaic virus (squash), and Soybean mosaic virus (bean).Conclusions: This study demonstrated the efficiency of NIb2F2/NIb3R in detection of potyviruses and revealed the extent of infections with diverse potyviruses species in the northwest part of Iran.

Background: P.atrosepticum is a commercially important pathogen. It causes blackleg in the field and soft rot of tubers after the harvest. This effect is due to secretion of depolymerases and other virulence factors by several mechanisms including T3SS Objectives: The effect of bacterial T3SS on Solanumtuberosum (S. tuberosum) varieties and its relationship with S. tuberosum resistance gene expression were studied.Materials and Methods: A P. atrosepticum HrpW gene was cloned, sequenced and constructed a phylogenic tree with some phytopathogen. The virulence properties of P. atrosepticum strains were investigated and then S. tuberosum varieties were tested for their sensitivity against P. atrosepticum. PR-5 and HIN genes copy-number for infiltrated S. tuberosum tubers were assessed. Results: The results show that infiltrated tubers of P. atrosepticum T3SS mutants were significantly more macerated than the wild type ones.Conclusions: PR-5 and HIN expression amounts were depended on bacterial T3SS function.