重组酶介导等温扩增技术检测疟原虫方法的建立和评价

Establishment and evaluation of a method for detection of Plasmodium by recombinase-aided amplification

目的 建立一种重组酶介导等温扩增技术(RAA)检测疟原虫的方法。方法 针对疟原虫属保守18S小亚基核糖体RNA(18S rRNA)基因设计特异性引物,筛选确定最佳引物对组合并建立疟原虫重组酶介导核酸等温扩增体系。通过该RAA体系检测疟原虫标准质粒、4种疟原虫[恶性疟原虫(Plasmodium falciparum, P.f)、间日疟原虫(P. vivax,P.v)、三日疟原虫(P. malaria, P.m)、卵形疟原虫(P. ovale, Po)]病原体以及其他6种输血传播寄生虫(婴儿利士曼原虫、杜氏利士曼原虫、刚地弓形虫、溶组织阿米巴虫、犬吉氏巴贝西虫、田鼠巴贝虫),以评估该体系的灵敏度和特异度。结果 筛选出一组扩增效果较好的引物对,整个RAA体系使用最佳引物对在37℃,20 min即可完成扩增,对标准质粒的检测下限为10-2copies/μL,恶性疟原虫、间日疟原虫和卵形疟原虫为102copies/μL,三日疟原虫为10 copies/μL。RAA检测体系未与其他输血传播寄生虫产生交叉反应,特异性良好。应用RAA方法检测外籍学生献血者标本,结果与荧光定量PCR结果一致。结论 成功建立了一种快速、简便和特异的疟原虫RAA检测方法,将会给血液筛查和一些基层偏远医院的临床检测提供很大帮助。

Objective To establish a recombinase-aided amplification(RAA) assay method for detection of Plasmodium species.Methods Plasmodium-specific primers are designed for the conserved 18S small subunit ribosomal RNA(18SrRNA) gene. Plasmodium standard plasmids, DNA of four Plasmodium pathogens, Plasmodium falciparum(P.f), P. vivax(P.v),P. malaria(P. m), P. ovale(P. o) and six other transfusion-transmitted parasites(Leishmania infantile, Leishmania durandii,Toxoplasma gondii, Amoeba histolytica, Babesia guisi, and Babesia microti) were used to assess the sensitivity and specificity ofthe RAA system.ResultsA set of primer pairs with good amplification effect were selected. The whole RAA system could beamplified at 37 ℃ for 20 minutes using the optimal primer pair. The detection limit of standard plasmids was 10-2copies/μL, Pf,Pv and Po was 102copies/μL, and Pm was 10 copies/μL. The RAA system did not cross-react with other transfusion-transmitted parasites and showed good specificity. The samples from foreign blood donors were tested by RAA, and the resultswere all negative.Conclusions A simple and rapid RAA detection method with good sensitivity and specificity has beenestablished, which will provide great help for blood donor screening and clinical detection in some remote areas.