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

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国家科技期刊平台
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中文摘要：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1h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.

外文标题：Clinical study of isothermal nucleic acid detection technology based on CRISPR Cas12a for highly sensitive diagnosis of Cryptococcus neoformans

外文摘要：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.

外文关键词：

Cryptococcus neoformansisothermal recombinase polymerase amplificationCRISPR Cas12aclin-ical diagnosis

作者：

叶辛、刘伊诺、张蕾、罗明明、李俊玉、张黎明、方文捷、刘馨遥、冉玉平、潘炜华、许斌、廖万清

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作者单位：

西安交通大学第一附属医院检验科,西安 710061

海军军医大学附属长征医院皮肤科,上海 200003

陕西省人民医院皮肤科,西安 710068

江西省肿瘤医院,南昌 330029

四川大学华西医院,成都 610044

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关键词：

新型隐球菌等温重组酶聚合酶扩增 CRISPR Cas12a 临床诊断

基金：

西安交通大学第一附属医院院级基金科技部重点研发计划国家自然科学基金国家自然科学基金国家自然科学基金上海市科委项目上海市科委项目

项目编号：

2021ZYTS-182022YFC250480382202543820722578210241620DZ227200021410750500

出版年：

2024

DOI：

10.16190/j.cnki.45-1211/r.2024.04.001

广西医科大学学报

广西医科大学

广西医科大学学报

CSTPCD

影响因子：0.788

ISSN：1005-930X

年,卷(期)：2024.41(4)

参考文献量18