Evaluation of a Loop-Mediated Isothermal Amplification Method as a Tool for Diagnosis of Infection by the Zoonotic Simian Malaria Parasite Plasmodium knowlesi
Evaluation of a Loop-Mediated Isothermal Amplification Method as a Tool for Diagnosis of Infection by the Zoonotic Simian Malaria Parasite Plasmodium knowlesi
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DOI：
10.1128/jcm.00331-10
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发表时间：
2010-07-01
影响因子：
9.4
通讯作者：
Igarashi, Ikuo
中科院分区：
文献类型：
--
作者：
Iseki, Hiroshi;Kawai, Satoru;Igarashi, Ikuo
环介导的等温扩增(LAMP)是一种在等温条件下快速扩增特异性高的靶DNA的新方法。它已被用作几种传染病的诊断工具，包括病毒、细菌和寄生虫病。在本研究中，我们建立了诺氏疟原虫感染的LAMP分子诊断方法(PkLAMP)，并评价了其敏感性、特异性和临床适用性。我们设计了三套针对物种特异性β-微管蛋白基因的PkLAMP引物。所设计的PkLAMP引物能特异地扩增诺氏假单胞菌的自体DNA提取液，其灵敏度是单一PCR方法的100倍。这些结果表明，我们的PkLAMP方法可以有效地区分诺氏疟原虫和其他疟疾寄生虫。为了评价活体材料应用的可行性，将PkLAMP与传统的套式聚合酶链式反应(NPCR)方法进行了比较，并对两种实验感染的猕猴的血液样本进行了镜检。这些研究表明，用PkLAMP可以比nPCR和显微镜更早地识别诺氏肺孢子虫感染。此外，以全血为模板的PkLAMP的检测性能与以基因组DNA为模板的PkLAMP的检测性能相同。这些结果表明，PkLAMP方法是流行地区诺氏肺孢子虫感染的一种有前景的分子诊断工具。
Loop-mediated isothermal amplification (LAMP) is a novel method that rapidly amplifies target DNA with high specificity under isothermal conditions. It has been applied as a diagnostic tool for several infectious diseases, including viral, bacterial, and parasitic diseases. In the present study, we developed a LAMP method for the molecular diagnosis of Plasmodium knowlesi infection (PkLAMP) and evaluated its sensitivity, specificity, and clinical applicability. We designed three sets of PkLAMP primers for the species-specific beta-tubulin gene. The primer sets for PkLAMP specifically amplified the autologous DNA extracts of P. knowlesi, and the sensitivity of the test was 100-fold that of single-PCR assay. These results indicate that our PkLAMP method can be used to efficiently distinguish between P. knowlesi and other malaria parasites. To evaluate the feasibility of using in vivo materials, comparisons of PkLAMP and the conventional nested PCR (nPCR) method and microscopic examination were made with blood samples from two experimentally infected monkeys. These studies showed that P. knowlesi infection can be identified much earlier with PkLAMP than with nPCR and microscopy. Moreover, the detection performance of PkLAMP using whole blood as the template was identical to that of PkLAMP when genomic DNA extracts were used. These results suggest that the PkLAMP method is a promising tool for molecular diagnosis of P. knowlesi infection in areas of endemicity.