Melioidosis is a bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei), posing a significant threat to public health. Rapid and accurate detection of B. pseudomallei is crucial for preventing and controlling melioidosis. However, identifying B. pseudomallei is challenging due to its high similarity to other species in the same genus. To address this issue, this study proposed a dual-target method that can specifically identify B. pseudomallei in less than 40 min. We analyzed 1722 B. pseudomallei genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish B. pseudomallei from its closely related species. Specifically, we selected two specific tags, LC1 and LC2, which we combined with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas12a) system and recombinase polymerase amplification (RPA) pre-amplification. Our analysis showed that the dual-target RPA-CRISPR/Cas12a assay has a sensitivity of approximately 0.2 copies/reaction and 10 fg genomic DNA for LC1, and 2 copies/reaction and 20 fg genomic DNA for LC2. Additionally, our method can accurately and rapidly detect B. pseudomallei in human blood and moist soil samples using the specific sequence tags mentioned above. In conclusion, the dual-target RPA-CRISPR/Cas12a method is a valuable tool for the rapid and accurate identification of B. pseudomallei in clinical and environmental samples, aiding in the prevention and control of melioidosis.
Clostridium perfringens ε-toxin (ETX) is the main toxin leading to enterotoxemia of sheep and goats and is classified as a potential biological weapon. In addition, no effective treatment drug is currently available in clinical practice for this toxin. We developed membrane-camouflaged nanoparticles (MNPs) with different membrane origins to neutralize ETX and protect the host from fatal ETX intoxication. We evaluated the safety and therapeutic efficacy of these MNPs in vitro and in vivo. Compared with membranes from karyocytes, such as Madin-Darby canine kidney (MDCK) cells and mouse neuroblastoma N2a cells (N2a cells), membrane from erythrocytes, which do not induce any immune response, are superior in safety. The protective ability of MNPs was evaluated by intravenous injection and lung delivery. We demonstrate that nebulized inhalation is as safe as intravenous injection and that both modalities can effectively protect mice against ETX. In particular, pulmonary delivery of nanoparticles more effectively treated the challenge of inhaled toxins than intravenously injected nanoparticles. Moreover, MNPs can alter the biological distribution of ETX among different organs in the body, and ETX was captured, neutralized and slowly delivered to the liver and spleen, where nanoparticles with ETX could be phagocytized and metabolized. This demonstrates how MNPs treat toxin infections in vivo. Finally, we injected the MNPs into mice in advance to find out whether MNPs can provide preventive protection, and the results showed that the long-cycle MNPs could provide at least a 3-day protection in mice. These findings demonstrate that MNPs provide safe and effective protection against ETX intoxication, provide new insights into membrane choices and delivery routes of nanoparticles, and new evidence of the ability of nanoparticles to provide preventive protection against infections. Graphical Abstract
Francisella tularensis is a dangerous pathogen that causes an extremely contagious zoonosis in humans named tularemia. Given its low-dose morbidity, the potential to be fatal, and aerosol spread, it is regarded as a severe threat to public health. The US Centers for Disease Control and Prevention (CDC) has classified it as a category A potential agent for bioterrorism and a Tier 1 Select Agent. Herein, we combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a system to select the F. tularensis target gene (TUL4), creating a two-pronged rapid and ultrasensitive diagnostic method for detecting F. tularensis. The real-time RPA (RT-RPA) assay detected F. tularensis within 10 min at a sensitivity of 5 copies/reaction, F. tularensis genomic DNA of 5 fg, and F. tularensis of 2 × 102 CFU/ml; the RPA-CRISPR/Cas12a assay detects F. tularensis within 40 min at a sensitivity of 0.5 copies/reaction, F. tularensis genomic DNA of 1 fg, and F. tularensis of 2 CFU/ml. Furthermore, the evaluation of specificity showed that both assays were highly specific to F. tularensis. More importantly, in a test of prepared simulated blood and sewage samples, the RT-RPA assay results were consistent with RT-PCR assay results, and the RPA-CRISPR/Cas12a assay could detect a minute amount of F. tularensis genomic DNA (2.5 fg). There was no nonspecific detection with blood samples and sewage samples, giving the tests a high practical application value. For example, in on-site and epidemic areas, the RT-RPA was used for rapid screening and the RPA-CRISPR/Cas12a assay was used for more accurate diagnosis.
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