Objective: 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 of 1 000, 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 1 000, 500, 200, 50, 10 parasites/μL with healthy volunteers' O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested.
Results: The Pf-F3/Pf-R3/crRNA2 combination, 2.5 μL as the addition amount of B buffer, 40 min as the RAA reaction time, 37 °C 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 containing 18S 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/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/μL.
Conclusions: The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.
［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/CRISPR-Cas12a 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1 000、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1 000、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、2.5 μL作为B buffer添加量、40 min作为RAA反应时间和37 °C 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/CRISPR-Cas12a 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6 d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/CRISPR-Cas12a法检测恶性疟原虫快速、敏感、特异, 有望用于恶性疟原虫快速检 测和风险监测。.
Keywords:
CRISPR-Cas12a; Detection efficiency;