Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.
Background Recent deep sequencing technologies have proven to be valuable resources to gain insights into the expression profiles of diverse tRNAs. However, despite these technologies, the association of tRNAs with diverse diseases has not been explored in depth because analytical tools are lacking. Results We developed a user-friendly tool, tRNA Expression Analysis Software Utilizing R for Easy use (tReasure), to analyze differentially expressed tRNAs (DEtRNAs) from deep sequencing data of small RNAs using R packages. tReasure can quantify individual mature tRNAs, isodecoders, and isoacceptors. By adopting stringent mapping strategies, tReasure supports the precise measurement of mature tRNA read counts. The whole analysis workflow for determining DEtRNAs (uploading FASTQ files, removing adapter sequences and poor-quality reads, mapping and quantifying tRNAs, filtering out low count tRNAs, determining DEtRNAs, and visualizing statistical analysis) can be performed with the tReasure package. Conclusions tReasure is an open-source software available for download at https://treasure.pmrc.re.kr and will be indispensable for users who have little experience with command-line software to explore the biological implication of tRNA expression.
Background Pyrazinamide (PZA) is often used as an add-on agent in the treatment of multidrug-resistant tuberculosis, regardless of phenotypic drug susceptibility testing (pDST) results. However, evaluating the effectiveness of PZA is challenging because of its low pH activity, which can result in unreliable pDST results. This study aimed to investigate the genomic characteristics associated with PZA resistance that can be used to develop genotypic DST. Materials and Methods A publicly available whole genome sequencing (WGS) dataset of 10,725 Mycobacterium tuberculosis complex genomes (3,326 phenotypically PZA-resistant and 7,399 phenotypically PZA-susceptible isolates) were analyzed. Results In total, 2,934 pncA non-silent mutations were identified in 2,880 isolates (26.9%). Detected mutations were found throughout the entire coding region of pncA in a scattered pattern, of which the most frequent mutation was p.Q10P (n = 278), followed by p.H57D (n = 167) and c.-11A>G (n = 122). The sensitivity and specificity of the group 1 or 2 mutations reported by the World Health Organization (WHO) mutational catalogue were 73.0% and 98.9%, respectively. We further identified 18 novel pncA mutations that were significantly associated with phenotypically PZA-resistant. In addition to these mutations, we identified 102 large deletions in the pncA gene, and all but two isolates were phenotypically resistant to PZA isolates. Notably, pncA deletions were mutually exclusive to pncA mutations, and more than half of the isolates with pncA large deletions belonged to the East Asian lineage (67.6%). The sensitivity, specificity, positive predictive value, and negative predictive value of the pooled variants (group 1 or 2 mutations, novel resistance-associated mutations, and large deletions of the pncA gene) were 79.0%, 98.9%, 97.0%, and 91.3%, respectively. The area under the curve (AUC) value for the pooled variants was significantly higher than the AUC value for the group 1 or 2 mutations ( P <0.001), indicating that the pooled variants have a better discriminative ability for predicting PZA resistance. Conclusion Using WGS, we found that the pncA mutations are scattered without specific mutational hotspots, and large deletions associated with PZA resistance are more common in the East Asian lineage of M. tuberculosis isolates. Our data also demonstrated the reliability of group 1 or 2 mutations presented in the WHO mutation catalogue and the need for further investigation on group 3 mutations, contributing to the evaluation of the current knowledge base on mutations associated with the PZA-resistant ...
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