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

目的:构建基于CRISPR Cas12a的高敏等温核酸检测方法,并评估其在支气管肺泡灌洗液及脑脊液中对于新型隐球菌的诊断能力。方法:利用NCBI Blast确定新型隐球菌保守序列,针对保守序列,设计等温重组酶聚合酶扩增引物及crRNA序列。利用新型隐球菌标准菌株及其他菌株(白念珠菌、热带念珠菌、光滑念珠菌、近平滑念珠菌、金黄色葡萄球菌、脑膜炎奈瑟菌、肺炎链球菌),提取其核酸,以评估方法的特异性及最低检测限。进一步收集临床确诊的10例肺部及10例中枢神经系统新型隐球菌感染患者的支气管肺泡灌洗液及脑脊液,以评估本研究构建的核酸诊断方法的临床检测性能。结果:共设计6组引物及crRNA组合,经筛选得到1组最佳组合。本研究构建的检测方法特异性高(与7种其他类型菌株无交叉反应),最低检测限达到5 copies/μL,整个检测流程速度快(在1 h内完成)。针对10例支气管肺泡灌洗液及10例脑脊液临床标本的阳性检出率均为100%。结论:本研究构建的新型核酸检测方法,具有高度的特异性和良好的检测限,在临床标本检测中同样具有优良检测性能,具备了较强的临床新型隐球菌核酸检测的应用潜力。

Objective:To construct a highly sensitive isothermal nucleic acid detection method based on CRISPR 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 targeting these conserved sequences.Nucleic acids were extracted from standard strains of Cryptococcus neoformans and other strains(Candida albicans,Candida tropicalis,Candida glabrata,Candida parapsilosis,Staphylococcus aureus,Neisseria meningitidis,Streptococcus pneumoniae) to evaluate the specificity and the lowest detection limit of the method.Furthermore,bronchoalveolar lavage fluid and cerebrospinal fluid samples from 10 clinically diagnosed cases of Cryptococcus neoformans infection in the lungs and central nervous system were collected 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 after screening.The detection method constructed in this study exhibited high specificity(no cross-reaction with 7other types of strains),a lowest detection limit of 5 copies/μL,and rapid detection process(completed within 1hour).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 specimen testing,and showing strong potential for clinical application in the detection of Cryptococcus neoformans nucleic acids.