机构：[1]Department of Ophthalmology,Shanghai General Hospital (Shanghai First People’s Hospital), Shanghai Jiao Tong University, School of Medicine, Shanghai[2]the National Clinical Research Center for Eye Diseases,Shanghai[3]the Shanghai Key Laboratory of Fundus Diseases,Shanghai[4]the Shanghai Engineering Center for Visual Science and Photomedicine,Shanghai[5]the Department of Hematology,Zhongnan Hospital of Wuhan University, Wuhan[6]the Gene Editing Center,School of Life Science and Technology, ShanghaiTech University, Shanghai[7]the Beijing Institute of Ophthalmology,Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, Beijing研究所眼科研究所首都医科大学附属北京同仁医院首都医科大学附属同仁医院[8]the Shenzhen Branch,Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rula Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China
Leber hereditary optic neuropathy (LHON) is the most common maternally inherited mitochondrial disease, with >90% of cases harboring one of three point variants (m.3460G>A, m.11778G>A, and m.14484T>C). Rapid and sensitive diagnosis of LHON variants is urgently needed for early diagnosis and timely treatment after onset, which is currently limited. Herein, we adapted the Cas12a-based DNA detection platform for LHON mitochondrial variant diagnosis. Single-strand guide CRISPR RNAs and enzymatic recombinase amplification primers were first screened, the CRISPR/Cas12a system was then optimized with restriction enzymes, and finally compared with Sanger sequencing and next -generation sequencing (NGS) in multicenter clinical samples. This approach can be completed within 30 minutes using only one drop of blood and could reach a sensitivity of 1% of heteroplasmy. Among the 182 multicenter clinical samples, the CRISPR/Cas12a detection system showed high consistency with Sanger sequencing and NGS in both specificity and sensitivity. Notably, a sample harboring a de novo 3.78% m.11778G>A variant detected by NGS, but not by Sanger sequencing, was successfully confirmed using the CRISPR/Cas12a assay, which proved the effectiveness of our method. Overall, our CRISPR/Cas12a detection system provides an alternative for rapid, convenient, and sensitive detection of LHON variants, exhibiting great potential for clinical practice. (J Mol Diagn 2023
基金：
National Natural Science Foundation of China [82171076, 81730026, 82101159, 82000906]; Science and Technology Commission of Shanghai Municipality [20Z11900400]; Shanghai Hospital Development Center [SHDC2020CR2040B, SHDC2020CR5014]; Shanghai Collaborative Innovation Center for Translational Medicine [TM202115PT]
第一作者机构：[1]Department of Ophthalmology,Shanghai General Hospital (Shanghai First People’s Hospital), Shanghai Jiao Tong University, School of Medicine, Shanghai[2]the National Clinical Research Center for Eye Diseases,Shanghai[3]the Shanghai Key Laboratory of Fundus Diseases,Shanghai
共同第一作者：
通讯作者：
通讯机构：[1]Department of Ophthalmology,Shanghai General Hospital (Shanghai First People’s Hospital), Shanghai Jiao Tong University, School of Medicine, Shanghai[2]the National Clinical Research Center for Eye Diseases,Shanghai[3]the Shanghai Key Laboratory of Fundus Diseases,Shanghai[6]the Gene Editing Center,School of Life Science and Technology, ShanghaiTech University, Shanghai[7]the Beijing Institute of Ophthalmology,Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, Beijing[8]the Shenzhen Branch,Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rula Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China[*1]Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, No. 7, Pengfei Rd., Dapeng District, Shenzhen 518120, China[*2]Shanghai General Hospital, School of Medicine, Shanghai Jiao Tong University, 100 Hai Ning Rd., Shanghai 200080, China.
推荐引用方式(GB/T 7714)：
Wan Xiaoling,Chen Jieqiong,Wu Yidong,et al.Rapid and Sensitive Diagnosis of Leber Hereditary Optic Neuropathy Variants Using Detection[J].JOURNAL OF MOLECULAR DIAGNOSTICS.2023,25(8):540-554.doi:10.1016/j.jmoldx.2023.04.006.
APA：
Wan, Xiaoling,Chen, Jieqiong,Wu, Yidong,Chen, Zhixuan,Liu, Yin...&Sun, Xiaodong.(2023).Rapid and Sensitive Diagnosis of Leber Hereditary Optic Neuropathy Variants Using Detection.JOURNAL OF MOLECULAR DIAGNOSTICS,25,(8)
MLA：
Wan, Xiaoling,et al."Rapid and Sensitive Diagnosis of Leber Hereditary Optic Neuropathy Variants Using Detection".JOURNAL OF MOLECULAR DIAGNOSTICS 25..8(2023):540-554
