The genus Cladophialophora comprises etiologic agents of disease in immunocompetent patients, ranging from mild cutaneous colonization to cerebral encephalitis, in addition to saprobic species. Due to the high degree of phenotypic similarity between closely related species of the genus, identification problems are imminent. In the present study, we described rapid and sensitive rolling circle amplification (RCA) method based on species-specific padlock probes targeted for the internal transcribed spacer regions of rDNA. ITS regions of 12 Cladophialophora species were sequenced, and subsequently, 10 specific padlock probes were designed for the detection of single nucleotide polymorphisms. The majority of circularizable padlock probes were designed based on single nucleotide polymorphisms (SNPs), while for C. bantiana, C. immunda and C. devriesii were characterized by two or more nucleotides. Individual species-specific probes correctly identified in all ten Cladophialophora species correctly by visualization on 1.2 % agarose gels used to verify specificity of probe-template binding; no cross-reactivity was observed. Simplicity, sensitivity, robustness and low costs provide RCA a distinct place among isothermal techniques for DNA diagnostics. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications.
In this study we reported the synthesis of L-phenyl alanine (Phe) & L-tyrosine (Tyr) Natural Amino acids coated iron oxide magnetic nanoparticles under one-pot and in situ reaction. Functionalized iron oxide magnetic nanoparticles were characterized by X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Vibrating Sample Magnetometer (VSM), Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) techniques. Cellular toxicity of amino acids coated iron oxide magnetic nanoparticles was also investigated on HEK-293 cell lines. Additionally, a hemolysis test of as prepared magnetic nanoparticles were performed. It was found that the synthesized Phe and Tyr coated magnetic nanoparticles (F@Phe NPs and F@Tyr NPs) were spherical in shape with an average size less than 25 nm, also the saturation magnetization (Ms) of the F@Phe NPs and F@Tyr NPs were about 30.02 and 58.23 emu/g, respectively, which was lower than those of bare FeO. The TGA results show that apart from this weight loss, the coated sample shows a weight loss of 5.48, and 6.88% respectively corresponding to loss of Tyr, and Phe which is coated on the FeO nanoparticles. At a high concentration, less than 2.92 and 3.13% hemolytic activity were observed for F@Phe NPs and F@Tyr NPs, respectively. The F@Phe NPs and F@Tyr NPs show the possibility of using this nanoparticles in the development of in vitro and in vivo pharmaceutical and biomedical fields due to do not possess a toxic effect, good ζ-potential and related small and narrow size distribution.
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