荧光定量PCR技术检测疟原虫方法初探  

A preliminary study on the detection of Plasmodium by fluorescence quantitative PCR

作　　者：邵雷[1] 朱姗姗[1] 胡文佳[1] 蒋昵真[1] 陈妍[1] 林红[1] SHAO Lei;ZHU Shanshan;HU Wenjia;JIANG Nizhen;CHEN Yan;LIN Hong(HongJiangsu Province Blood Center,Nanjing 210042,China)

机构地区：[1]江苏省血液中心,江苏南京210042

出　　处：《中国输血杂志》2020年第11期1144-1147,共4页Chinese Journal of Blood Transfusion

基　　金：江苏省卫计委高层次卫生人才“六个一工程”项目(LGY2016045);江苏省科技厅社会发展面上项目(BE2019755)。

摘　　要：目的探索荧光定量PCR技术在献血者疟原虫检测中的应用。方法参照文献设计针对疟原虫18S小亚基核糖体RNA (18S rRNA)基因序列的通用引物和探针并构建标准质粒;将标准质粒和4种疟原虫阳性标本进行梯度倍比稀释后进行荧光定量PCR检测,建立符合本实验室条件的实验体系,与巢式PCR方法进行灵敏度比较,并对47例献血者进行疟原虫筛查。结果应用荧光定量PCR技术检测标准质粒的检测下限是10-1copy/μL,用于恶性疟原虫(Plasmodium falciparum)、间日疟原虫(P.vivax)、三日疟原虫(P.malaria)和卵形疟原虫(P.ovale)阳性标本检测,其下限均为1 copy/μL,且整个PCR程序仅用时1 h 59 min;应用荧光定量PCR技术检测47例献血者全血标本,均为阴性;应用巢式PCR技术检测间日疟原虫和三日疟原虫的检测下限为102copy/μL,检测灵敏度明显低于荧光定量PCR检测方法。结论荧光定量PCR检测方法具有操作简单、快速、灵敏、经济等特点,通过针对本实验室的特定实验优化,更加适用于献血者疟原虫的快速大量筛查。Objective To explore the application of fluorescence quantitative PCR in Plasmodium detection on blood donors.Methods With reference to the literature, specific primers and probes for the 18 S small subunit ribosomal RNA(rRNA) gene sequence of Plasmodium were designed and the standard plasmid was constructed. Standard plasmid and four plasmodium positive samples were diluted by gradient dilution and detected by fluorescence quantitative PCR. An experimental system in line with the laboratory conditions was established and compared with Nested PCR method in terms of sensitivity, and 47 blood donors were screened for Plasmodium.Results The detection limit of standard plasmids by fluorescence quantitative PCR was 10-1 copy/μL, while the lower limit for Plasmodium falciparum, P. vivax, P. malariae and P. ovale was 1 copy/μL, and the whole PCR procedure only took 1 h 59 min. The samples of 47 whole blood donors were tested by fluorescence quantitative PCR, and the results were all negative. However, the detection threshold of Nested PCR for P. vivax and P. malariae were 102 copy/μL, and the detection sensitivity was significantly lower than that of fluorescence quantitative PCR.Conclusion Fluorescence quantitative PCR method is simple, rapid, sensitive and economical, which is more suitable for rapid and mass screening of blood donor plasmodium after specific experimental optimization in our laboratory.

关 键 词：疟原虫 荧光定量PCR 18S核糖体RNA 巢式PCR 献血者 

分 类 号：R457.1[医药卫生—治疗学] R382.31[医药卫生—临床医学]