Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a “sample-in-answer-out” single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.

Escherichia coli sequence type 405 is an emerging antibiotic-resistant clonal group associated with the global dissemination of extended-spectrum β-lactamase-producing E. coli. In this study, we report the genome assembly and characterization of a uropathogenic E. coli ST405 strain, SZESBLEC201, based on long and short reads obtained from the Nanopore and Illumina sequencing platforms, respectively. Whole-genome sequencing revealed that SZESBLEC201 harbors a 5,020,403 bp chromosome and three plasmids, namely, pSZESBLEC201-1, pSZESBLEC201-2, and pSZESBLEC201-3. pSZESBLEC201-1 (111,621 bp) belongs to the IncFIA-FIB type and harbors blaCTX-M-15. However, this plasmid does not harbor conjugative transfer-associated genes, rendering pSZESBLEC201-1 unable to be conjugatively transferred. pSZESBLEC201-2 (95,138 bp) is a phage-like plasmid that shows a strong genome synteny with Escherichia phage P1 but with the absence of mobile genetic elements and some regulatory genes. pSZESBLEC201-3 (92,865 bp) belongs to the IncI1 type and carries blaCTX-M-24. In contrast to pSZESBLEC201-1, pSZESBLEC201-3 retains its full active conjugation machinery and can be transferred via conjugation. The genetic features of the genome show that the SZESBLEC201 has a unique virulence pattern compared with genetically similar strains found in the same country (China). The plasmid backbones exhibit a high degree of similarity to those of geographically distant isolates, highlighting the global spread of blaCTX-M genes and the genome plasticity of this clonal group. The coexistence of two blaCTX-M variants in the same strain increases the risk of the emergence of new blaCTX-M variants. Further studies on phage-like plasmids are necessary to provide insights into their biological activities and clinical significance.

Bedaquiline has been widely used as a part of combination dosage regimens for the treatment of multidrug-resistant tuberculosis (MDR-TB) patients with limited options. Although the effectiveness and safety of bedaquiline have been demonstrated in clinical trials, limited studies have investigated the significant pharmacokinetics and the impact of genotype on bedaquiline disposition.