A universal all-in-one RPA-Cas12a strategy with de novo autodesigner and its application in on-site ultrasensitive detection of DNA and RNA viruses.

Revolutionary all-in-one RPA-CRISPR assays are rapidly becoming the most sought-after tools for point-of-care testing (POCT) due to their high sensitivity and ease of use. Despite the availability of one-pot methods for specific targets, the development of more efficient methods for new targets remains a significant challenge. In this study, we present a rapid and universal approach to establishing an all-in-one RPA-Cas12a method CORDSv2 based on rational balancing amplification and Cas12a cleavage, which achieves ultrasensitive detection of several targets, including SARS-CoV-2, ASFV, HPV16, and HPV18. CORDSv2 demonstrates a limit of detection (LOD) of 0.6 cp/µL and 100% sensitivity for SARS-CoV-2, comparable to qPCR. Combining with our portable device(hippo-CORDS), it has a visual detection LOD of 6 cp/µL and a sensitivity up to 100% for SARS-CoV-2 and 97% for Ct<35 ASFV samples, surpassing most one-pot visual methods. To simplify and accelerate the process for new targets, we also develop a de novo autodesigner by which the optimal couples of primers and crRNA can be selected rapidly. As a universal all-in-one RPA-CRISPR method for on-site testing, CORDSv2 becomes an attractive choice for rapid and accurate diagnosis in resource-limited settings.

Changes of gut mycobiota in the third trimester of pregnant women with preeclampsia.

OBJECTIVE: Studies recently acknowledged that the fungal community in the gut plays an important role in many inflammatory diseases of noninfectious origin. but the role of gut fungi in the pathogenesis of preeclampsia (PE) remains unknown. METHODS: We performed a case-case-control study to compare the gut mycobiota of PE, pregnancy with chronic hypertension (PCH), and the normal group (Normal pregnancy group without any underlying disease) by internal transcribed spacer sequencing. In addition, LC/MS was used to explore the relationship between the fecal metabolites and gut mycobiota. RESULTS: Compared with the PCH and the normal group, α diversity (represented the species abundance in a single sample) of mycobiota were lower in the PE group, but there was no statistically significant difference among these three groups. However, Linear discriminant analysis Effect Size (LEfSe) analysis found 3 differentially abundant fungal taxa in PE group when compared with the normal group. The gut metabolites of PE patients were significantly different from PCH and the normal group. Choline metabolism molecule glycerophosphocholine was the most discriminant metabolite between PE and the normal group. Correlation analysis found that Candida spp.was positively correlated with glycerophosphocholine which increased in PE. CONCLUSION: We found that gut mycobiota changed in the third trimester of pregnant women with preeclampsia.