A rapid and ultra-sensitive dual readout platform for Klebsiella pneumoniae detection based on RPA-CRISPR/Cas12a.

The bacterium Klebsiella pneumoniae (Kp) was the primary pathogen of hospital-acquired infection, but the current detection method could not rapidly and conveniently identify Kp. Recombinase polymerase amplification (RPA) was a fast and convenient isothermal amplification technology, and the clustered regularly interspaced short palindromic repeats (CRISPR) system could rapidly amplify the signal of RPA and improve its limit of detection (LOD). In this study, we designed three pairs of RPA primers for the rcsA gene of Kp, amplified the RPA signal through single-strand DNA reporter cleavage by CRISPR/Cas12a, and finally analyzed the cleavage signal using fluorescence detection (FD) and lateral flow test strips (LFTS). Our results indicated that the RPA-CRISPR/Cas12a platform could specifically identify Kp from eleven common clinical pathogens. The LOD of FD and LFTS were 1 fg/µL and 10 fg/µL, respectively. In clinical sample testing, the RPA-CRISPR/Cas12a platform was consistent with the culture method and qPCR method, and its sensitivity and specificity were 100% (16/16) and 100% (9/9), respectively. With the advantages of detection speed, simplicity, and accuracy, the RPA-CRISPR/Cas12a platform was expected to be a convenient tool for the early clinical detection of Kp.

Cysteine protease of Clonorchis sinensis alleviates DSS-induced colitis in mice.

BACKGROUND: Currently, inflammatory bowel disease (IBD) has become a global chronic idiopathic disease with ever-rising morbidity and prevalence. Accumulating evidence supports the IBD-hygiene hypothesis that helminths and their derivatives have potential therapeutic value for IBD. Clonorchis sinensis (C. sinensis) mainly elicit Th2/Treg-dominated immune responses to maintain long-term parasitism in the host. This study aimed to evaluate the therapeutic effects of cysteine protease (CsCP) and adult crude antigen (CsCA) of C. sinensis, and C. sinensis (Cs) infection on DSS-induced colitis mice. METHODS: BALB/c mice were given 5% DSS daily for 7 days to induce colitis. During this period, mice were treated with rCsCP, CsCA or dexamethasone (DXM) every day, or Cs infection which was established in advance. Changes in body weight, disease activity index (DAI), colon lengths, macroscopic scores, histopathological findings, myeloperoxidase (MPO) activity levels, regulatory T cell (Treg) subset levels, colon gene expression levels, serum cytokine levels, and biochemical indexes were measured. RESULTS: Compared with Cs infection, rCsCP and CsCA alleviated the disease activity of acute colitis more significant without causing abnormal blood biochemical indexes. In comparison, rCsCP was superior to CsCA in attenuating colonic pathological symptoms, enhancing the proportion of Treg cells in spleens and mesenteric lymph nodes, and improving the secretion of inflammatory-related cytokines (e.g., IL-2, IL-4, IL-10 and IL-13) in serum. Combined with RNA-seq data, it was revealed that CsCA might up-regulate the genes related to C-type lectin receptor and intestinal mucosal repair related signal pathways (e.g., Cd209d, F13a1 and Cckbr) to reduce colon inflammation and benefit intestinal mucosal repair. Dissimilarly, rCsCP ameliorated colitis mainly through stimulating innate immunity, such as Toll like receptor (TLR) signaling pathway, down-regulating the expression of inflammatory cytokines (e.g., IL-12b, IL-23r and IL-7), thereby restraining the differentiation of Th1/Th17 cells. CONCLUSIONS: Both rCsCP and CsCA showed good therapeutic effects on the treatment of acute colitis, but rCsCP is a better choice. rCsCP is a safe, effective, readily available and promising therapeutic agent against IBD mainly by activating innate immunity and regulating the IL-12/IL-23r axis.