Silica-coated magnetic particles for efficient RNA extraction for SARS-CoV-2 detection.

Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.

Swarming motility in Bradyrhizobium japonicum.

Flagellar-driven bacterial motility is an important trait for colonization of natural environments. Bradyrhizobium japonicum is a soil species that possesses two different flagellar systems: one subpolar and the other lateral, each with a filament formed by a different set of flagellins. While synthesis of subpolar flagellins is constitutive, translation of lateral flagellins was detected in rhizobia grown with l-arabinose, but not with d-mannitol as sole carbon source, independently of whether bacteria were in liquid or semisolid medium. We characterized swarming of B. japonicum in semisolid medium and found that this motility was faster with l-arabinose than with d-mannitol. By using mutants with deletions in each flagellin set, we evaluated the contribution of each flagellum system to swarming in semisolid culture media, and in soil. Mutants devoid of either of the flagella were affected in swarming in culture media, with this impairment being stronger for mutants without lateral flagella. In sterile soil at 100% or 80% field capacity, flagellar-driven motility of mutants able to swim but impaired in swarming was similar to wild type, indicating that swimming was the predominant movement here.

[Frequency of dermatophytes in a sample of cats in the urban area of Gran Mendoza, Argentina]. / Frecuencia de dermatofitos en una muestra de felinos del área urbana del Gran Mendoza, Argentina.

BACKGROUND: The cat, considered the main reservoir of Microsporum canis, lives in urban areas, and also plays an important role in the emergence of dermatomycoses. AIMS: To determine and analyse the frequency of zoonotic dermatophytes in a sample of cats in an urban area of the Gran Mendoza region. METHODS: The animals selected were household cats and cats less than one year old that came from shelters and kennels from urban areas in the Gran Mendoza region. A total of 45 samples from cats with and without dermatological lesions were analysed. These samples were collected through skin scraping, hair removal and Mackenzie brush, respectively. Direct observation was made with KOH and glycerol after heat exposure. Samples were cultured on Sabouraud and Lactrimel agar slants with chloramphenicol and cycloheximide for 30 days. RESULTS: The frequency of dermatophytes isolated in this preliminary study was 13.3%. There were not statistically significant differences by source, age, sex, race or dermatological condition. Zoonotic dermatophytes were found in 2 household cats out of the 21 that had direct contact with children or the elderly. M. canis was isolated in 83.3% cases. CONCLUSIONS: The frequency of isolation of zoonotic dermatophytes in the sample of cats in an urban area of the Gran Mendoza region was 13.3%, a value higher than expected. M. canis was the most isolated species.

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean.

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate.