套式PCR扩增特定SSrRNA基因片段诊断间日疟及混合感染的研究

DETECTION OF PLASMODIUM VIVAX AND MIXED INFECTION BY AMPLIFYING SPECIFIC SSrRNA GENE WITH NESTED POLYMERASE CHAIN REACTION

根据间日疟原虫（Ｐ．ｖ．）小亚单位核糖体核糖核酸（ＳＳｒＲＮＡ）基因序列，设计并合成两对特异引物，采用套式聚合酶链反应（ＰＣＲ）技术，扩增出一条长１２０个碱基对（ｂｐ）的间日疟原虫ＳＳｒＲＮＡ基因特定片段，并用于云南间日疟患者的血样检测。结果表明：该系统特异性强，除间日疟原虫外，恶性疟原虫（Ｐ．ｆ．）、三日疟原虫（Ｐ．ｍ．）及正常人血ＤＮＡ样本均无此扩增带出现。该系统检测原虫的敏感度为０．１个疟原虫／ｌμｌ血，大大高于常规镜检。在进行间日疟检测中，该系统与镜检的阳性符合率为１００％，更重要的是发现镜检漏诊的４例Ｐ．ｖ．和Ｐ．ｆ．的混合感染病例。鉴于该系统具有特异、敏感、稳定、简便的特点和鉴别疟原虫种类、判定混合感染等优点，故对疟疾的诊断。

Two pairs of oligonucleotide primers were designed and synthesized based on small subunit ribosomal RNA gene of plasmodium vivax , and blood samples obtained from malaria vivax patients in Yunnan were analyzed by nested polymerase chain reaction (PCR ). The results showed that a 120 base pairs(bp) DNA fragment of P.vivax was amplified. No amplification was observed with P. falciparum , P. malariae and human DNA. The nested PCR could detect parasitemia to an extent as low as 0.1 parasite/μl. The positive concordance rate between nested PCR and microscopy was 100% in detecting P.vivax . In addition, 4 cases of mixed plasmodium infection were found by PCR. It is suggested that this method is specific, sensitive, stable and has a great potential in identifying different plasmodium species and detecting mixed infections. Thus, PCR assay provides a powerful tool in study of malaria diagnosis and epidemiology.