广西医科大学学报2024，Vol.41Issue(4)：483-488,6.DOI:10.16190/j.cnki.45-1211/r.2024.04.001
基于CRISPR Cas12a的等温核酸检测技术用于新型隐球菌高敏诊断的临床研究
Clinical study of isothermal nucleic acid detection technology based on CRISPR Cas12a for highly sensitive diagnosis of Cryptococcus neoformans
摘要
Abstract
Objective:To construct a highly sensitive isothermal nucleic acid detection method based on CRIS-PR Cas12a and evaluate its diagnostic ability for Cryptococcus neoformans in bronchoalveolar lavage fluid and cerebrospinal fluid.Methods:Conserved sequences of Cryptococcus neoformans were determined using NCBI Blast,and isothermal recombinase polymerase amplification primers and crRNA sequences were designed target-ing these conserved sequences.Nucleic acids were extracted from standard strains of Cryptococcus neoformans and other strains(Candida albicans,Candida tropicalis,Candida glabrata,Candida parapsilosis,Staphylococ-cus aureus,Neisseria meningitidis,Streptococcus pneumoniae)to evaluate the specificity and the lowest detec-tion limit of the method.Furthermore,bronchoalveolar lavage fluid and cerebrospinal fluid samples from 10 clini-cally diagnosed cases of Cryptococcus neoformans infection in the lungs and central nervous system were collect-ed to evaluate the clinical detection performance of the nucleic acid diagnostic method constructed in this study.Results:A total of 6 primer and crRNA combinations were designed,and 1 optimal combination was selected af-ter screening.The detection method constructed in this study exhibited high specificity(no cross-reaction with 7 other types of strains),a lowest detection limit of 5 copies/μL,and rapid detection process(completed within 1 hour).The positive detection rates for bronchoalveolar lavage fluid and cerebrospinal fluid clinical specimens from 10 cases each were 100%.Conclusion:The novel nucleic acid detection method constructed in this study has high specificity and a good detection limit,demonstrating excellent detection performance in clinical speci-men testing,and showing strong potential for clinical application in the detection of Cryptococcus neoformans nu-cleic acids.关键词
新型隐球菌/等温重组酶聚合酶扩增/CRISPR Cas12a/临床诊断Key words
Cryptococcus neoformans/isothermal recombinase polymerase amplification/CRISPR Cas12a/clin-ical diagnosis分类
医药卫生引用本文复制引用
叶辛,潘炜华,许斌,廖万清,刘伊诺,张蕾,罗明明,李俊玉,张黎明,方文捷,刘馨遥,冉玉平..基于CRISPR Cas12a的等温核酸检测技术用于新型隐球菌高敏诊断的临床研究[J].广西医科大学学报,2024,41(4):483-488,6.基金项目
西安交通大学第一附属医院院级基金资助项目(No.2021ZYTS-18) （No.2021ZYTS-18）
科技部重点研发项目(No.2022YFC2504803) （No.2022YFC2504803）
国家自然科学基金资助项目(No.82202543 （）
No.82072257 （）
No.82102416) （）
上海市科委基金资助项目(No.20DZ2272000 （）
No.21410750500) （）
