Specific detection of gut pathogens for one-pot chip based on RPA-CRISPR/Cas12a.

BACKGROUND: There are billions of bacteria in the intestine, most of which are harmless and play important roles in humans. Although only a very small number of bacteria can cause diseases, once the pathogenic bacteria are ingested into the body and multiply in large quantities, it can lead to inflammatory diseases in the intestines and even other organs. Although polymerase chain reaction can specifically detect bacterial nucleic acid. However, the demand for temperature cycling limits its portability. Therefore, it is hoped to establish a high-throughput, highly specific and portable detection platform for directly detecting nucleic acid of intestinal pathogens. RESULTS: Herein, a one-pot chip based on RPA-CRCISPR/Cas12a platform was developed. The chip is the same size as a glass slide and allows detection at the same temperature. Multiple samples could be detected simultaneously on the one chip, achieved high-throughput detection and improved the integration of detection. The specific recognition of CRISPR/Cas12a avoided the influence of non-specific amplification of RPA and enhanced the specificity of the analysis. At the same time, the one-pot chip avoided secondary contamination when the lid was opened during the analysis process. And the bacterial concentration showed good linearity at 102-108 cfu mL-1. The limit of detection could be as low as 0.43 cfu mL-1. This method has been successfully used to detect pollution samples. It can provide a reliable platform for early screening of gastrointestinal and other inflammatory diseases. SIGNIFICANCE: The one-pot chip based on the RPA-CRISPR/Cas12a platform established can directly detect the nucleic acid of intestinal pathogens, with portability and specificity. It is worth noting that the platform has good programmability, can be used for other target detection by changing crRNA and RPA primers, it can achieve multi sample detection on the one chip.

Sensitive detection of microRNA by dynamic light scattering based on DNAzyme walker-mediated AuNPs self-assembly.

As an important biomarker, microRNAs (miRNAs) play an important role in gene expression, and their detection has attracted increasing attention. In this study, a DNAzyme walker that could provide power to perform autonomous movement was designed. Based on the continuous mechanical motion characteristics of DNAzyme walker, a miRNA detection strategy for the self-assembly of AuNPs induced by the hairpin probe-guided DNAzyme walker "enzyme cleavage and walk" was established. In this strategy, DNAzyme walker continuously cleaved and walked on the hairpin probe on the surface of AuNPs to induce the continuous shedding of some segments of the hairpin probe. The remaining hairpin sequences on the surface of the AuNP pair with each other, causing the nanoparticles to self-assemble. This strategy uses the autonomous movement mechanism of DNAzyme walker to improve reaction efficiency and avoid the problem of using expensive and easily degradable proteases. Secondly, using dynamic light scattering technology as the signal output system, ultra-sensitive detection with a detection limit of 3.6 fM is achieved. In addition, this strategy has been successfully used to analyze target miRNAs in cancer cell samples.