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OACSTPCD

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.