Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis
Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis
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DOI：
10.1128/jcm.02117-06
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发表时间：
2007-08-01
影响因子：
9.4
通讯作者：
Tsuboi, Takafumi
中科院分区：
文献类型：
--
作者：
Han, Eun-Taek;Watanabe, Risa;Tsuboi, Takafumi
建立了一种新的核酸扩增方法——环介导等温扩增（LAMP），用于临床检测恶性疟原虫、间日疟原虫、疟疾疟原虫和卵形疟原虫。我们将LAMP的敏感性和特异性与显微镜检查和巢式PCR的结果进行比较。LAMP的检测限（分析灵敏度）为疟疾疟原虫和卵形疟原虫10个拷贝，疟原虫、恶性疟原虫和间日疟原虫100个拷贝。LAMP在68份镜检阳性血样中检出67份疟原虫（敏感性98.5%），在53份镜检阴性血样中检出3份疟原虫（特异性94.3%），与巢式PCR结果吻合较好。LAMP反应平均在26分钟内得到结果，疟原虫检测32分钟，间日疟原虫检测31分钟，疟疾疟原虫检测35分钟，卵形疟原虫检测36分钟。因此，与镜检结果相比，LAMP具有相似的灵敏度和更高的特异性，并且LAMP在更短的周转时间内获得与巢式PCR相似的结果。由于LAMP可以用一种简单的技术进行，即以热处理过的血液为模板，在水浴中进行反应，并使用荧光染料用肉眼检查结果，因此LAMP可以为疟疾流行地区的临床实验室和疟疾诊所提供一种简单可靠的常规筛查疟疾寄生虫的方法。
Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovate. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovate and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovate. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.