套式聚合酶链式反应诊断恶性疟原虫及混合感染的研究

DETECTION OF PLASMODIUM FALCIPARUM AND MIXED INFECTION BY POLYMERASE CHAIN REACTION

为了建立一种特异、敏感、简便的疟疾诊断方法，设计并合成两对针对恶性疟原虫（Ｐ．ｆ．）小亚单位核糖体核糖核酸（ＳＳＵｒＲＮＡ）基因特异的引物，采用套式聚合酶链式反应（ＰＣＲ）技术，扩增恶性疟原虫ＳＳＵｒＲＮＡ基因特定片段。利用该系统诊断云南及海南疟疾患者血样，并随机选取扩增产物进行克隆测序鉴定。结果显示，恶性疟原虫模板均扩增出长度为２０５ｂｐ的特定基因片段，经Ｔ－载体克隆测序与恶性疟原虫ＳＳＵｒＲＮＡ特定基因片段序列完全符合。该系统特异性强，除恶性疟原虫外，间日疟原虫（Ｐ．ｖ．）、三日疟原虫（Ｐ．ｍ．）、卵形疟原虫（Ｐ．ｏ．）、正常人血ＤＮＡ样本及空白对照均无此扩增带出现。该系统检测原虫的敏感度为１．５个原虫／μｌ，大大高于常规镜检。６１份Ｐ．ｆ．镜检阳性标本在进行ＰＣＲ检测时亦均呈阳性，同时联合应用套式ＰＣＲ扩增间日疟原虫ＳＳＵｒＲＮＡ特定基因片段的检测系统，发现６１例恶性疟病人中２３例是Ｐ．ｆ．和Ｐ．ｖ．混合感染。该检测系统具有特异、敏感、稳定、简便的特点，对疟疾的诊断。

In order to establish a specific, sensitive and simple diagnosis method for malaria, two pairs of oligonucleotide primers were designed and synthesized according to the sequence of small subunit ribosomal RNA gene of Plasmodium falciparum . A specific DNA fragment was amplified by polymerase chain reaction. Blood samples collected from malaria falciparum patients in Yunnan and Hainan were analyzed by this diagnostic system, 2 PCR products which were chosen randomly were cloned and their sequences were determined. The results showed that a 205 bp gene fragment was amplified from all templates of Plasmodium falciparum whose sequence is as same as a part of small subunit ribosomal RNA gene of Plasmodium falciparum . No positive bands were observed with Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and human DNA. The nested PCR could detect parasitemia to an extent as low as 1.5 paratites/μl。 The positive concordance rate between nested PCR and microscopy is 100% in detecting 61 cases of P.f. infection. In addition, we found 23 patients with mixed infection by PCR assay detecting P.f. and P.v. simultaneously while microscopists failed to do so. It is suggested that PCR method is specific, sensitive, stable and simple in malaria diagnosis and detection of different plasmodial species mixed infections.