Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis
Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis
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DOI：
10.1128/jcm.42.3.1214-1219.2004
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发表时间：
2004-03-01
影响因子：
9.4
通讯作者：
Chezzi, C
中科院分区：
文献类型：
--
作者：
Perandin, F;Manca, N;Chezzi, C
设计了一种基于taqman的实时PCR定性检测方法，用于检测三种疟疾寄生虫——恶性疟原虫、卵形疟原虫和间日疟原虫。该方法使用了122份来自疟疾流行地区旅行并出现疟疾样症状和发烧的患者的全血样本，并对其进行了评估。将该试验与常规显微镜和已建立的巢式pcr试验进行比较。通过对所有阳性样本的PCR产物进行测序，以及与刚地弓形虫和婴儿利什曼原虫DNA缺乏交叉反应性，证实了新检测方法的特异性。实时荧光定量PCR检测结果显示，恶性疟原虫、间日疟原虫和卵形疟原虫的检出限（分析灵敏度）分别为0.7、4和1.5只/株。Real-time PCR与巢式PCR一样，通过显微镜检查发现了物种鉴定的错误，并发现了恶性疟原虫和卵形疟原虫混合感染的存在。实时PCR可以在2小时内产生结果，不需要PCR后处理，减少样品处理，并最大限度地降低污染风险。因此，该检测方法可以很容易地用于常规疟疾诊断检测。该技术的临床应用价值有待进一步研究。
A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites-Plasmodium falciparum, P. ovale, and P. vivax-was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/mul for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.