多重qPCR检测疟原虫方法的建立及应用评价  

Development and application of a multiplex qPCR assay for the detection of four Plasmodium species

作　　者：张家祺 徐昌平[1] 阮卫[1] 王笑笑[1] 陈华良[1] 张轩[1] ZHANG Jia-qi;XU Chang-ping;RUAN Wei;WANG Xiao-xiao;CHEN Hua-liang;ZHANG Xuan(Zhejiang Provincial Center for Disease Control and Prevention,Zhejiang 310051,China)

机构地区：[1]浙江省疾病预防控制中心,浙江310051

出　　处：《中国卫生检验杂志》2023年第4期393-396,共4页Chinese Journal of Health Laboratory Technology

基　　金：浙江省医药卫生科研项目(2020PY038)。

摘　　要：目的 建立一种能检测4种疟原虫多重qPCR(multiplex quantitative real-time polymerase chain reaction)方法,并对其进行临床应用评价。方法 以4种疟原虫18S rRNA基因作为靶序列,在保守区域设计双侧扩增引物,根据4种疟原虫特异性区域分别设计荧光探针,建立多重qPCR检测方法,并测试其特异性、敏感性,同时应用该方法对疟疾或疑似疟疾的临床血样进行检测。结果 建立检测4种疟原虫多重qPCR方法具有良好的特异性与敏感性,检测下限可达10拷贝/μl。该方法对浙江省2020年—2021年115份疟疾或疑似疟疾的临床血样进行检测,结果与疟疾分子诊断参考方法巢式PCR一致,临床特异度与敏感度均为100%。结论 本研究所建立的检测4种疟原虫多重qPCR方法简单快速、特异性好、敏感性高,可应用于疟疾临床病原诊断和参比实验室样本复核。Objective This paper aims to develop a multiplex quantitative real-time polymerase chain reaction(qPCR) assay for the detection of four Plasmodium species and evaluate its clinical application. Methods Using the 18S rRNA genes of four Plasmodium species as target sequences, bilateral amplification primers were designed in the conserved regions, and fluorescent probes were designed according to the specific regions of four plasmodium species respectively. A multiplex qPCR detection method was established, and its specificity and sensitivity were tested. At the same time, the method was applied to detect clinical blood samples of malaria or suspected malaria. Results The multiplex qPCR assay for the detection of four Plasmodium species had good specificity and sensitivity, and the limit of detection was up to 10 copies/μl. 115 clinical blood samples of malaria or suspected malaria in Zhejiang Province from 2020 to 2021 were tested, and the results were consistent with the reference method for molecular diagnosis of malaria, nested PCR, and the clinical specificity and sensitivity were both 100%. Conclusion The multiplex qPCR assay developed in this study is simple, rapid, specific and sensitive for the detection of four Plasmodium species, and can be applied to clinical malaria pathogenic diagnosis and evaluation of treatment effects.

关 键 词：疟原虫 荧光定量PCR TAQMAN探针 

分 类 号：R446[医药卫生—诊断学]