荧光重组酶聚合酶扩增/CRISPR-Cas12a快速检测恶性疟原虫方法的建立与初步评价  

Establishment and preliminary evaluation of a fluorescent recombinase-aided amplification/CRISPR-Cas12a system for rapid detection of Plasmodium falciparum

作　　者：黄维益 韦华贵 王春芳[2] 王俊利 陈丽莹 陈伟忠 刘亚群[4] 郑玉忠 林敏[1,4] HUANG Weiyi;WEI Huagui;WANG Chunfang;WANG Junli;CHEN Liying;CHEN Weizhong;LIU Yaqun;ZHENG Yu-zhong;LIN Min(School of Laboratory Medicine,Youjiang Medical University for Nationalities,Baise,Guangxi 533000,China;Department of Laboratory Medicine,The Affiliated Hospital of Youjiang Medical University for Nationalities,Baise,Guangxi 533000,China;Department of Laboratory Medicine,Chaozhou People’s Hospital Affiliated to Shantou University,Chaozhou,Guangdong 521000,China;College of Life Science and Food Engineering,Hanshan Normal University,Chaozhou,Guangdong 521000,China)

机构地区：[1]右江民族医学院医学检验学院,广西百色533000 [2]右江民族医学院附属医院检验科,广西百色533000 [3]汕头大学附属潮州市人民医院检验科,广东潮州521000 [4]韩山师范学院生命科学与食品工程学院,广东潮州521000

出　　处：《中国血吸虫病防治杂志》2023年第1期38-43,67,共7页Chinese Journal of Schistosomiasis Control

基　　金：广西自然科学基金重点项目(2019JJD140052);广西自然科学基金面上项目(2020JJA140656);广东省教育厅重点项目(2019-GDXK-0031);广东省粤东食药资源功能物质与治未病研究重点实验室(1202B1212040015);广东省潮州市科技计划项目(202102GY15);2021年度广东省潮州市卫生健康局科研项目(2021013)。

摘　　要：目的建立一种基于重组酶聚合酶扩增(recombinase-aided amplification,RAA)和CRISPR-Cas12a系统的荧光快速检测恶性疟原虫方法,并对其检测效能进行初步评价。方法选择恶性疟原虫18S核糖体RNA(rRNA)基因为靶序列,设计和合成3组RAA引物及CRISPR来源RNA(crRNA),选择最佳组合并优化反应条件,建立荧光RAA/CRISPRCas12a检测方法。构建包括靶标区的恶性疟原虫3D7株18S rRNA基因质粒,将质粒浓度分别稀释成1000、100、10、1拷贝数/μL进行荧光RAA/CRISPR-Cas12a反应,评价荧光RAA/CRISPR-Cas12a法检测敏感度;分别以间日疟原虫、三日疟原虫、卵形疟原虫、乙型肝炎病毒、人类免疫缺陷病毒、梅毒螺旋体基因组DNA为模板,进行荧光RAA/CRISPR-Cas12a反应,评价荧光RAA/CRISPR-Cas12a法检测特异度。分别采用荧光RAA/CRISPR-Cas12a法和巢式PCR法对50份疟疾临床样本进行检测,比较两种方法检测一致性。体外培养恶性疟原虫3D7株,经培养后获得的培养物采用健康人O型红细胞悬液稀释成1000、500、200、50、10个/μL等不同虫密度血液样本,以荧光RAA/CRISPR-Cas12a法进行检测,评价检测效果。结果选择Pf-F3/Pf-R3/crRNA2作为引物组合、2.5μL作为B buffer添加量、40 min作为RAA反应时间和37℃作为CRISPR-Cas12a反应温度建立荧光RAA/CRISPR-Cas12a法,其可检出浓度为1拷贝数/μL的含恶性疟原虫3D7株18S rRNA基因的质粒;该法检测恶性疟原虫可见荧光信号,但检测卵形疟原虫、三日疟原虫、间日疟原虫及阴性对照均无荧光信号,且与梅毒螺旋体、人类免疫缺陷病毒、乙型肝炎病毒等其他病原体亦无交叉反应。采用荧光RAA/CRISPRCas12a法和巢式PCR法分别检测50份疟疾临床样本,两种方法检测结果完全一致(kappa值=1.0,P<0.001)。恶性疟原虫3D7株经6d体外培养,获得10 mL培养物,采用荧光RAA/CRISPR-Cas12a法检测稀释后的临床样本,得到最低检测限为50个疟原虫/μL。结论荧光RAA/CObjective To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification(RAA)and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.Methods The 18S ribosomal RNA(rRNA)gene of P.falciparum was selected as the target sequence,and three pairs of RAA primers and CRISPR-derived RNA(crRNA)were designed and synthesized.The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system.The plasmid containing 18S rRNA gene of the P.falciparum strain 3D7 was generated,and diluted into concentrations of1000,100,10,1 copy/μL for the fluorescent RAA/CRISPR-Cas12a assay,and its sensitivity was evaluated.The genomic DNA from P.vivax,P.malariae,P.ovum,hepatitis B virus,human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay,and its specificity was evaluated.Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay,and the consistency between two assays was compared.In addition,P.falciparum strain 3D7 was cultured in vitro.Then,the culture was diluted into blood samples with parasite densities of 1000,500,200,50,10 parasites/μL with healthy volunteers’O-positive red blood cells for the RAA/CRISPRCas12a assay,and the detection efficiency was tested.Results The Pf-F3/Pf-R3/crRNA2 combination,2.5μL as the addition amount of B buffer,40 min as the RAA reaction time,37℃as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system.Such a system was effective to detect the plasmid containing18S rRNA gene of the P.falciparum strain 3D7 at a concentration of 1 copy/μL,and presented fluorescent signals for detection of P.falciparum,but failed to detect P.ovum,P.malariae,P.vivax,T.pallidum,hepatitis B virus or human immunodeficiency virus.The fluorescent RAA/CRIS

关 键 词：恶性疟原虫 重组酶聚合酶等温扩增 CRISPR-Cas12a 检测效能 

分 类 号：R382.31[医药卫生—医学寄生虫学]