背景:肠道中有数十亿细菌,其中大多数是无害的,对人类起着重要作用。虽然只有极少数的细菌可以引起疾病,一旦致病菌被摄入体内并大量繁殖,它可以导致肠道甚至其他器官的炎症性疾病。虽然聚合酶链反应可以特异性检测细菌核酸。然而,对温度循环的需求限制了其便携性。因此,希望建立高通量,高特异性和便携式检测平台,可直接检测肠道病原体的核酸。
结果:这里,开发了基于RPA-CRCISPR/Cas12a平台的一锅芯片。芯片的大小与载玻片相同,并且允许在相同温度下进行检测。可以在一个芯片上同时检测多个样本,实现了高通量检测,提高了检测的集成度。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,一锅芯片避免了分析过程中打开盖子时的二次污染。细菌浓度在102-108cfumL-1范围内呈良好的线性关系。检测限可以低至0.43cfumL-1。该方法已成功用于污染样品的检测。可为胃肠道等炎症性疾病的早期筛查提供可靠平台。
结论:基于RPA-CRISPR/Cas12a平台建立的一锅芯片可以直接检测肠道病原菌的核酸,具有可移植性和特异性。值得注意的是,该平台具有良好的可编程性,可通过改变crRNA和RPA引物用于其他靶标检测,它可以在一个芯片上实现多样本检测。
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.
CONCLUSIONS: 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.