结核病疫情和耐药趋势严峻，结核性脑膜炎是最严重的结核病类型，病死率、病残率高，但目前的诊断方法存在费时费力、敏感性不够高、价格高昂等不足。我们及之前的研究发现rpoB基因已成为结核及耐药诊治的潜在新靶点。基于CRISPR/Cas基因编辑II类Cas核酸酶具有的“附带切割”特性，本课题拟建立一种结核菌特异性CRISPR/Cas12b高灵敏、简便核酸检测方法：选择rpoB核心突变区序列，设计对应的crRNA，等温扩增目的基因，借助CRISPR/Cas12b基因编辑激活的Cas12b切割单链DNA探针，通过检测荧光信号的变化而实现对结核菌核酸的高灵敏检测。之后，将该基因编辑系统采用侧向流层析技术进行核酸检测，由此获得对包括利福平耐药信息在内的敏感、简便的结核菌核酸检测试纸，进一步评估其在结核性脑膜炎诊断中的作用。旨在从基因编辑水平上建立结核菌灵敏、简便的检测方法，为结核病的诊治提供新策略。

Tuberculosis is the leading killer of infectious diseases worldwide, the epidemic situation of tuberculosis and the trend of drug resistance are severe. Tuberculous meningitis is the most serious type of tuberculosis with high fatality rate and disability rate.However,the current diagnostic methods are time-consuming, laborious,insufficiently sensitive or expensive. Our and previous studies have found that rpoB gene has become a potential new target for the diagnosis and treatment of tuberculosis and drug resistance. Based on the nonspecific “collateral cleavage” activity in class II Cas proteins of CRISPR/Cas gene editing system, this project intends to establish a kind of CRISPR/Cas12b mediated, highly sensitive, simple nucleic acid assay targeting for Mycobacterium tuberculosis: rpoB specific core area mutations will be chosen, corresponding CRISPR RNA (crRNA) will be designed, after isothermal amplification, CRISPR/Cas12b gene editing will specifically recognize the target gene and activate Cas12b nonspecific "collateral cleavage" nuclease activity to cut single-strand DNA probes, the high sensitive nucleic acid testing for tuberculosis will be realized by detecting the change of fluorescent signal. Subsequently,the M.tuberculosis gene editing system will be used for nucleic acid testing by lateral flow chromatography assay, thus a sensitive and simple M.tuberculosis nucleic acid test strip including rifampicin resistance information will be obtained. Then, its role in the diagnosis of tuberculous meningitis will be further evaluated. The aim of this study is to establish a sensitive and simple detection method for tuberculosis at the gene editing level and to provide a novel strategy for the diagnosis and treatment of tuberculosis including tuberculous meningitis.