Aim: The aim of this study was designing a LAMP method for the rapid detection of Brucella and development of a sensitive quantitative-LAMP (Q-LAMP) assay for quantification of brucellosis. Methods and Results: In this study for the LAMP detection of the causative agent of brucellosis, we used specifically designed primers to target the omp25 conserved gene of Brucella spp. The sensitivity of the LAMP method was evaluated by preparing serial tenfold dilution of omp25 gene containing plasmid followed by performing the LAMP reaction. To improve the assay as a quantitative test, LAMP products in the serial dilution were evaluated by Loopamp real-time turbidimeter system and then standard curve was generated by plotting time threshold values against log of copy number. The assay specificity was evaluated using Brucella genomic DNA and a panel containing genomes of 11 gram-positive and gramnegative organisms. The LAMP assay was highly specific and no amplification products were observed from the non-Brucella organisms. The test sensitivity for visual detection of turbidity or fluorescent colour change and also agarose gel electrophoresis was 560 ng and 5Á6 ng, respectively. The lower limit of detection was 17 copies of the gene that could be detected in 50 min. Conclusions: The results of this study indicated that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of Brucella spp. that may improve diagnostic potential in clinical laboratories. Significance and Impact of the Study: The LAMP assay because of the simplicity and low cost can be preferred to other molecular methods in the diagnosis of infectious diseases.
Introduction Acinetobacter baumannii are nosocomial bacteria that are responsible for outbreaks and severe infections in hospitalized patients globally. The major target of this study was the characterization of virulence determinants and biofilm formation of A. baumannii isolates from hospitalized patients. Methods In total, 100 A. baumannii were collected from three hospitals in Tehran, Iran, 2017-2018. The isolates were assessed using phenotypic and genotypic methods and then screened for virulence factor encoding genes such as plcN and lasB using conventional polymerase chain reaction. Furthermore, bacterial biofilm formation, motility and hemolytic and proteolytic activities were assessed. Results Of 100 A. baumannii isolates, 20 isolates included plcN and four isolates included lasB using PCR assay. Overall, 21 isolates were negative for biofilm formation while 45, 20 and 14 of the total isolates were reported as weak, moderate and strong biofilm producers, respectively. All isolates were positive for bap genes using PCR. Moreover, 35 isolates were motile on Luria-Bertani media, 47 isolates were αhemolytic on Brucella blood agar media and all isolates displayed proteolytic activity. Conclusions Healthcare-associated infections with A. baumannii are a major concern, importantly due to their potency to acquire virulence factor genes. Therefore, shedding light in the discovery of new antimicrobial and/or therapeutic agents against virulent A. baumannii strains seem to be necessary.
A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals and transgenic plants. Also, it has been reported that Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has the capability of expressing heterologous genes. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein. The oligosaccharide structures of their glycoproteins are similar to those of mammals with N-linked galactose, and sialic acid residues. For a variety of reasons, including the glycosylation patterns and the secondary structures of some of these proteins, synthesis in eukaryotic system is highly preferable. In addition, formation of native disulfide bonds in complex eukaryotic proteins is tremendously important. In the present study, we tried to express the tPA (tissue plasminogen activator) gene in L. tarentolae. This protein is a thrombolytic agent with 527 amino acid residues. tPA possesses serine-protease activity, with 35 cysteine residues that participate in the formation of 17 disulfide bonds. We have used an expression cassette, including the alpha intergenic regions of Leishmania major and two sites at the 3'- and 5'-ends, for homologous recombination in L. tarentolae, in addition to antibiotic-resistant genes. Southern-blot analysis showed that the human tPA gene had been inserted into the genome of the parasite. The expression of the tPA at the mRNA and protein levels was confirmed. It was shown that the expressed tPA in this system was 70 i.u. (international units)/ml of culture media, which is much higher than levels reported previously in other systems.
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