Sequencing the genomes of the circulating SARS-CoV-2 strains is the only way to monitor the viral spread and evolution of the virus. Two different approaches, namely, tiling multiplex PCR and sequence hybridization by bait capture, are commonly used to fulfill this task.
A variety of methods have been established in order to optimize the accessibility of DNA originating from Bacillus anthracis cells and endospores to facilitate highly sensitive molecular diagnostics. However, most endospore lysis techniques have not been evaluated in respect to their quantitative proficiencies. Here, we started by systematically assessing the efficiencies of 20 DNA extraction kits for vegetative B. anthracis cells. Of these, the Epicentre MasterPure kit gave the best DNA yields and quality suitable for further genomic analysis. Yet, none of the kits tested were able to extract reasonable quantities of DNA from cores of the endospores. Thus, we developed a mechanical endospore lysis protocol, facilitating the extraction of high-quality DNA. Transmission electron microscopy or the labelling of spores with the indicator dye propidium monoazide was utilized to assess lysis efficiency. Finally, the yield and quality of genomic spore DNA were quantified by PCR and they were found to be dependent on lysis matrix composition, instrumental parameters, and the method used for subsequent DNA purification. Our final standardized lysis and DNA extraction protocol allows for the quantitative detection of low levels (<50 CFU/mL) of B. anthracis endospores and it is suitable for direct quantification, even under resource-limited field conditions, where culturing is not an option.
is a pathogen that causes gastroenteritis in humans. Because of its low-temperature-dependent insecticidal activity, it can oscillate between invertebrates and mammals as host organisms. The insecticidal activity of strain W22703 is associated with a pathogenicity island of 19 kb (Tc-PAI ), which carries regulators and genes encoding the toxin complex (Tc). The island also harbors four phage-related and highly conserved genes of unknown functions, which are polycistronically transcribed. Two open reading frames showed significant homologies to holins and endolysins and exhibited lytic activity in cells upon overexpression. When a set of strains was tested in an equivalent manner, highly diverse susceptibilities to lysis were observed, and some strains were resistant to lysis. If cell lysis occurred (as demonstrated by membrane staining), it was more pronounced when two accessory elements of the cassette coding for an i-spanin and an o-spanin were included in the overexpression construct. The pore-forming function of the putative holin, HolY, was demonstrated by complementation of the lysis defect of a phage λ S holin mutant. In experiments performed with membrane preparations, ElyY exhibited high specificity for W22703 peptidoglycan, with a cleavage activity resembling that of lysozyme. Although the functionality of the lysis cassette from Tc-PAI was demonstrated in this study, its biological role remains to be elucidated. The knowledge of how pathogens survive in the environment is pivotal for our understanding of bacterial virulence. The insecticidal and nematocidal activity of spp., by which the bacteria gain access to nutrients and thus improve their environmental fitness, is conferred by the toxin complex (Tc) encoded on a highly conserved pathogenicity island termed Tc-PAI While the regulators and the toxin subunits of the island had been characterized in some detail, the role of phage-related genes within the island remained to be elucidated. Here, we demonstrate that this cassette encodes a holin, an endolysin, and two spanins that, at least upon overexpression, lyse strains.
Aim Rapid detection of biological agents in biodefense is critical for operational, tactical and strategic levels as well as for medical countermeasures. Yersinia pestis, Francisella tularensis, and Bacillus anthracis are high priority agents of biological warfare or bioterrorism and many response forces use lateral flow assays (LFAs) for their detection. Several companies produce these assays, which offer results in short time and are easy to use. Despite their importance, only few publications on the limits of detection (LOD) for LFAs are available. Most of these studies used inactivated bacteria or risk group‐2 strains. As the inactivation process in previous studies might have affected the tests’ performances, it was our aim in this study to determine and compare the LOD of several commercially available LFAs using viable risk group‐3 strains. Methods and Results Lateral flow assays from four different companies for the detection of following bacteria were evaluated: Y. pestis, F. tularensis and B. anthracis spores. Two independent quantification methods for each target organism were applied, in order to ensure high quantification accuracy. LODs varied greatly between tests and organisms and ranged between 104 for Y. pestis‐tests and as high as >109 for one B. anthracis‐test. Conclusion This work precisely determined the LODs of LFAs from four commercial suppliers. The herein determined LODs differed from results of previous studies. This illustrates the need for using accurately quantified viable risk group 3‐strains for determining such LODs. Significance and Impact of the Study Our work bridges an important knowledge gap with regard to LFA LOD. The LODs determined in this study will facilitate better assessment of LFA‐results. They illustrate that a negative LFA result is not suited to exclude the presence of the respective agent in the analyzed sample.
We report detection of Lassa virus and Crimean-Congo hemorrhagic fever virus infections in the area of Bamako, the capital of Mali. Our investigation found 2 cases of infection with each of these viruses. These results show the potential for both of these viruses to be endemic to Mali.
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