CRISPR-Cas13a辅助RAA快速检测金黄色葡萄球菌的研究  

Rapid detection of Staphylococcus aureus by CRISPR-Cas13a combined with recombinase aided amplification(RAA)

作　　者：苏璇 葛以跃[2] 张倩 朱小娟[2] 陈银[2] 吴涛[2] 乔乔 赵康辰[2] 吴斌[2] 王祥喜[3] 庞正 朱凤才[2] 崔仑标[2] SU Xuan;GE Yi-yue;ZHANG Qian;ZHU Xiao-juan;CHEN Yin;WU Tao;QIAO Qiao;ZHAO Kang-chen;WU Bin;WANG Xiang-xi;PANG Zheng;ZHU Feng-cai;CUI Lun-biao(College of Pharmacy,Nankai University,Tianjin,China 300000;NHC Key Laboratory of Enteric Pathogen Microbiology,Jiangsu Provincial Center for Disease Control and Prevention;Institute of Biophysics,ChineseA-cademy of Sciences;Tianjin International Biomedical Joint Research Institute)

机构地区：[1]南开大学药学院,天津300000 [2]国家卫生健康委员会肠道病原微生物重点实验室,江苏省疾病预防控制中心 [3]中国科学院生物物理研究所 [4]天津国际生物医药联合研究院

出　　处：《中国病原生物学杂志》2020年第3期253-258,共6页Journal of Pathogen Biology

基　　金：国家科技重大专项(No.2017ZX10302301-004);江苏省社会发展项目(No.BE2019761)。

摘　　要：目的建立一种金黄色葡萄球菌快速分子检测技术。方法根据金黄色葡萄球菌耐热核酸酶基因(nuc)保守区序列设计合成特异性引物,通过对反应条件进行优化,建立金黄色葡萄球菌重组酶介导的等温扩增技术(Recombi-naseaidedamplification,RAA);表达纯化CRISPR-Cas13a蛋白,设计特异的crRNA(CRISPR RNA),以crRNA引导CRISPR-Cas13a蛋白对RAA产物进行检测;对优化的方法进行灵敏性和特异性评价,同时采用该方法与real-timePCR法对食品标本中的金黄色葡萄球菌进行检测,评价方法的一致性。结果CRISPR-Cas13a辅助RAA检测金黄色葡萄球菌的灵敏度为101 CFU/ml,高于real-timePCR,约102 CFU/ml,检测时间仅需30min,与其他食源性致病菌无交叉反应;该方法和real-timePCR检测80份食品样品的阳性率均为8.75%,具有高度一致性(Kappa=1,P>0.05)。结论建立的CRISPR-Cas13a辅助RAA方法具有简便、快速、灵敏、特异等优点,为金黄色葡萄球菌的检测提供了新的技术手段。Objective To establish a technique for rapid molecular detection of Staphylococcus aureus.Methods Specific primers were designed and synthesized according to the sequence of the conserved region of the S.aureus thermonuclease gene(nuc).Recombinase-aided amplification(RAA)was performed by optimizing reaction conditions.CRISPR-Cas13a was expressed and purified.After specific crRNA(CRISPR RNA)was designed and synthesized,RAA products were detected with CRISPR-Cas13a,which was guided by crRNA.The sensitivity and specificity of the optimized method were also evaluated.At the same time,the technique and real-time PCR were both used to detect S.aureusin food samples,and their consistency was evaluated.Results The minimum detection limit of CRISPR-Cas13a combined with RAA with respect to S.aureus was 10~1 CFU/mL,which was more sensitive than real-time PCR(10~2 CFU/mL).The bacterium at a concentration of 101 CFU/mL was detected within 30 min,and templates at a higher concentration took less time.There was no cross-reaction with other food-borne pathogens.In 80 food samples,CRISPR-Cas13a combined with RAA and real-time PCR both detected the bacterium at a rate of 8.75%(Kappa=1,P>0.05),which indicated that the technique is highly consistent with real-time PCR.Conclusion As established in this study,CRISPR-Cas13a combined with RAA has the advantages of simplicity,rapidity,sensitivity,and specificity and provides a new method for detecting S.aureus.

关 键 词：RAA CRISPR-Cas13a 金黄色葡萄球菌 快速分子检测 

分 类 号：R378.11[医药卫生—病原生物学]