针对副溶血性弧菌toxR基因的RPA-CRISPR/Cas13a荧光快速检测方法的建立及应用
Establishment and application of RPA-CRISPR/Cas13a fluorescent rapid assay targeting Vibrio parahaemolyticus toxR gene
侯雅超 1邢微微 2王亚楠 1刘新萍 3董优优 3周光 4陈昌国5
作者信息
- 1. 河北北方学院医学检验学院,河北张家口 075000;解放军总医院第六医学中心检验科,北京 100048;军事科学院军事医学研究院军事认知与脑科学研究所,北京 100850
- 2. 军事科学院军事医学研究院军事认知与脑科学研究所,北京 100850
- 3. 解放军总医院第六医学中心检验科,北京 100048
- 4. 解放军总医院第一医学中心检验科,北京 100853
- 5. 河北北方学院医学检验学院,河北张家口 075000;解放军总医院第六医学中心检验科,北京 100048
- 折叠
摘要
目的 探讨并建立非专业实验室场景下恒温、快速及简便的检测副溶血性弧菌的方法.方法 本研究针对副溶血性弧菌toxR基因设计特异性引物和成簇规律间隔短回文重复序列(CRISPR)RNA(crRNA),建立基于重组酶聚合酶扩增(RPA)结合CRISPR及其相关蛋白13a(CRISPR-Cas13a)的反应体系,采用自配十二烷基硫酸钠(SDS)核酸快速提取试剂提取样本全基因组,并结合荧光法实现检测结果的可视化判读.结果 利用副溶血性弧菌菌株ATCC 17802和其他3种非副溶血性弧菌(溶藻弧菌、河流弧菌和梅氏弧菌)以及3种临床上常见腹泻致病菌(金黄色葡萄球菌、大肠埃希菌和铜绿假单胞菌)对RPA-CRISPR/Cas13a荧光法的特异性进行验证,结果显示特异性为100.00％;对副溶血性弧菌基因组DNA进行倍比稀释并检测,该方法最低检出限为102 copies/μl.最后,将建立的方法应用于野生型副溶血性弧菌检测,检测结果与TaqMan定量聚合酶链式反应(TaqMan-qPCR)检测结果及基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)鉴定结果一致.结论 本研究建立了一种针对toxR基因的RPA-CRISPR/Cas13a检测方法,具有简便、快速、特异性强、结果判读可视化等优点,为非专业实验室场景下的副溶血性弧菌快速检测提供了较好工具.
Abstract
OBJECTIVE To explore a constant,rapid and simple method for detection of Vibrio parahemolyticus in non-specialized laboratory settings.METHODS Specific primers and clustered regular interval short palindrome re-peating sequence(CRISPR)RNA(crRNA)were designed in targeting toxR gene of V.parahemolyticus so as to establish a reaction system based on recombinase polymerase amplification(RPA)and its related protein 13a(CRISPR-Cas13a).The whole genome was extracted from the samples by sodium dodecyl sulfate(SDS)nucleic acid rapid extraction reagent,and the visualized interpretation of the detection results was carried out by fluores-cence method.RESULTS The specificity of the RPA-CRISPR/Cas13a fluorescence method was verified by using V.parahemolyticus ATCC 17802 strains and 3 species of non-V.parahemolyticus(Vibrio alginolyticus,Vibrio fluvialis and Vibrio metschnikovi)as well as 3 common species of pathogens causing diarrhea(Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa),and the result showed that the specificity was 100.00％.The genome DNA of the V.parahemolyticus was subjected to serial dilutions and detected,and the minimum de-tectable limit of the method was 102 copies/μl.The established methods was applied for detection of wild type Ⅴ.parahemolyticus,and the detection result was consistent with the result of TaqMan quantitative polymerase chain reaction(TaqMan-qPCR)and the result of matrix-assisted laser desorption ionization-time-of-flight(MALDI-TOF)mass spectrometry.CONCLUSION The established RPA-CRISPR/Cas13a detection method targeting the toxR gene is simple,rapid and has high specificity and visualized interpretation of result,and it facilitates the rapid de-tection of V.parahemolyticus in non-specialized laboratory settings.
关键词
副溶血性弧菌/重组酶聚合酶扩增/成簇的规律间隔短回文重复序列及其相关蛋白13a/快速检测/可视化Key words
Vibrio parahemolyticus/Recombinase polymerase amplification/Clustered regular interval short pal-indrome repeating sequence and its related protein 13a/Rapid detection/Visualization引用本文复制引用
基金项目
国家自然科学基金资助项目(81401311)
出版年
2024
NETL
NSTL
万方数据