Trung Ngo Tat, Son Le Huu Phuc, Hien Trinh Xuan, Quyen Dao Thanh, Bang Mai Hong, Song Le Huu
Centre for Genetics Consultation and Cancer Screening (CGC), Hanoi, Vietnam.
Vietnamese-German Center for Medical Research (VG-CARE), Hanoi, Vietnam.
BMC Infect Dis. 2022 May 4;22(1):429. doi: 10.1186/s12879-022-07363-w.
Loop isothermal amplification (LAMP) has recently been proposed as a point-of-care diagnostic tool to detect acute infectious pathogens; however, this technique embeds risk of generating false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen's DNA targets complimerntary to its cognate RNA, afterward acquiring the collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs.
The MetA gene of N. meningitidis was selected as target to optimize the LAMP reaction, whereas pseudo-dilution series of N. meningitidis gemonics DNA was used to establish the detection limit of LAMP/Cas12a combination assay. The diagnostic performance of established LAMP/Cas12a combination assay was validated in comparation with standard real-time PCR on 51 CSF samples (14 N. meningitidis confirmed patients and 37 control subjects).
In relevant biochemical conditions, CRISPR-Cas12a and LAMP can work synchronously to accurately identify genetics materials of Nesseria menitigistis at the level 40 copies/reaction less than 2 h.
In properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.
环介导等温扩增技术（LAMP）最近被提议作为一种即时诊断工具来检测急性感染性病原体；然而，该技术存在产生假阳性结果的风险。而CRISPR/Cas12a能够准确识别特定序列，可与同源RNA传感器形成复合物，并切割与其同源RNA互补的病原体DNA靶点，随后获得附带活性以无差别地切割附近的非靶点片段。因此，如果反应中存在相关的荧光淬灭核酸探针，探针的非特异性切割会释放荧光并建立诊断读数。
选择脑膜炎奈瑟菌的MetA基因作为靶点来优化LAMP反应，而使用脑膜炎奈瑟菌基因组DNA的假稀释系列来确定LAMP/Cas12a联合检测的检测限。在51份脑脊液样本（14例确诊为脑膜炎奈瑟菌的患者和37例对照受试者）上，将建立的LAMP/Cas12a联合检测的诊断性能与标准实时PCR进行比较验证。
在相关生化条件下，CRISPR-Cas12a和LAMP可同步工作，在不到2小时内准确鉴定出反应水平低至40拷贝/反应的脑膜炎奈瑟菌遗传物质。
在适当优化的条件下，CRISPR-Cas12a系统有助于减少假阳性结果，从而提高LAMP检测的特异性。
