复式PCR检测恶性疟原虫与间日疟原虫的研究

目的： 建立在同一次扩增中即可鉴别患者感染的疟原虫虫种的复式ＰＣＲ检测方法。方法： 根据恶性疟原虫（Ｐ．ｆ．）中度重复基因序列ｐＢＲＫ１－１４ 和间日疟原虫（Ｐ．ｖ．）线粒体细胞色素Ｃ氧化酶基因ＣＯＩＩＩ合成引物，采用经优化的ＰＣＲ反应体系， 对疟原虫ＤＮＡ模板进行扩增。结果： Ｐ．ｆ．与Ｐ．ｖ．分别被扩增出２０６ 和３７０ ｂｐ 大小的ＤＮＡ片段，与人白细胞ＤＮＡ 无交叉；用该反应体系至少可检测出原虫血症为５×１０－ ７的Ｐ．ｆ．感染和１．０２×１０－ ６ Ｐ．ｖ．感染； 自云南疟疾流行区采集的７８３份滤纸干血滴样本中， 复式ＰＣＲ法阳性检出率为８５．８％ ， 误诊率为０， 漏诊率为０．１％ ， 而镜检法依次分别为８４．９％ 、３．１％ 和１．０％ ， 两者符合率为９５．８％ 。结论： 本复式ＰＣＲ检测疟原虫较镜检敏感、特异， 适用于我国恶性疟与间日疟混合流行区的疟疾诊断、流行病学调查、药物的疗效考核和献血员的筛选等。

STUDIES ON DETECTION OF PLASMODIUM FALCIPARUM AND PLASMODIUM VIVAX IN BLOOD SAMPLES BY MULTIPLEX POLYMERASE CHAIN REACTION

AIM: To establish a sensitive and specific PCR-based method to detect Plasmodium falciparum and P. vivax in blood samples in a single amplification reaction. METHODS: Malaria parasite DNA in blood was amplified by the multiplex polymerase chain reaction using two sets of primers derived from the P. f. moderately-repetitive DNA sequence and COIII gene of P.v. RESULTS: A 206-bp product for P. f. and a 370-bp product for P.v. were amplified by multiplex PCR, being able to detect parasitemia level as low as 5 x 10(-7) for P. f. and 1.02 x 10(-6) for P. v. and having no cross-reaction with human leucocyte DNA. A total of 783 blood samples on the filter paper collected from patients attending to malaria clinics in malaria endemic areas were detected. The positive rate of multiplex PCR was 85.8%, the misdiagnosis rate was 0, and the under-diagnosis rate was 0.1%, while these three rates of microscopic examination were 84.9%, 3.1% and 1.0%, respectively. The concordance between the two methods was 95.8%. CONCLUSION: The multiplex PCR method made the malaria detection more sensitive and specific than the microscopic examination and should be suitable for the diagnosis of malaria in mixed endemic areas, large-scale epidemiological studies, follow-up of drug treatment and donor blood screening.