4种疟原虫核酸通用型微滴式数字PCR检测方法的建立

Establishment of droplet digital PCR assay for the detection of four Plasmodium species

目的 建立疟原虫微滴式数字PCR(droplet digital PCR,ddPCR)检测方法。方法 根据疟原虫18S rRNA基因保守序列设计特异性引物和TaqMan探针,用于ddPCR方法建立。优化dd PCR的退火温度、引物和探针浓度后,比较ddPCR和实时荧光定量PCR(Real-time Quantitative PCR,qPCR)灵敏度和重复性,并对4种疟原虫阳性样本进行检测。结果 建立的ddPCR方法特异性良好,灵敏度可达到2.3拷贝/μl,重复性良好。13份经qPCR检测的阳性样本,ddPCR检测也均为阳性,正确率达100%;5份经一个疗程青蒿素为基础的联合疗法治疗后的疟疾病人样本,qPCR检测均为阴性,dd PCR检测发现其中1份阳性(3.8拷贝/μl)。结论 建立的ddPCR能够高度敏感、特异地检测人类4种疟原虫核酸。

Objective To establish a droplet digital PCR(ddPCR)method for the detection of four human Plasmodium species.Methods The primers and probe were designed for ddPCR according to 18S rRNA gene after verifying its specificity.The annealing temperature,primer,probe concentration of ddPCR were optimized simultaneously.The sensitivity and repeatability of ddPCR was compared with qPCR,and the samples were tested.Results The ddPCR had a specificity of 100%and a sensitivity of 2.3 copies/μl,and had a good reproducibility.Among the 13 blood samples with confirmed diagnosis of malaria by qPCR,13 were detected positive by ddPCR.Among the 5 blood samples from patients who were diagnosed of malaria and treated by a complete course of an ACT,one sample were detected positive by ddPCR.Conclusion The ddPCR is of high sensitivity and specificity,and could be used for the detection of four human Plasmodium species.