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. 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 10-10 cfu mL. The limit of detection could be as low as 0.43 cfu mL. 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. 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.

中文翻译：

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基于RPA-CRISPR/Cas12a的一锅芯片特异性检测肠道病原体


肠道内有数十亿细菌，其中大部分是无害的，但对人类发挥着重要作用。虽然只有极少数细菌会引起疾病，但致病菌一旦摄入体内并大量繁殖，就会导致肠道甚至其他器官的炎症性疾病。虽然聚合酶链式反应可以特异性检测细菌核酸。然而，温度循环的需求限制了其便携性。因此，希望建立一个高通量、高特异性、便携式的直接检测肠道病原体核酸的检测平台。在此，开发了一种基于RPA-CRCISPR/Cas12a平台的一锅式芯片。该芯片与载玻片大小相同，并且允许在相同温度下进行检测。可在一块芯片上同时检测多个样品，实现高通量检测，提高检测的集成度。 CRISPR/Cas12a的特异性识别避免了RPA非特异性扩增的影响，增强了分析的特异性。同时，一锅式芯片避免了分析过程中开盖时的二次污染。细菌浓度在 10-10 cfu mL 范围内表现出良好的线性。检测限可低至 0.43 cfu mL。该方法已成功用于污染样品的检测。可为胃肠道及其他炎症性疾病的早期筛查提供可靠的平台。建立的基于RPA-CRISPR/Cas12a平台的一锅芯片可以直接检测肠道病原体的核酸，具有便携性和特异性。 值得注意的是，该平台具有良好的可编程性，通过改变crRNA和RPA引物可用于其他目标检测，可在一块芯片上实现多样本检测。