Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria.
Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria.
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DOI：
10.1371/journal.pone.0013733
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发表时间：
2010-10-29
期刊：
影响因子：
3.7
通讯作者：
Udhayakumar V
中科院分区：
文献类型：
--
作者：
Lucchi NW;Demas A;Narayanan J;Sumari D;Kabanywanyi A;Kachur SP;Barnwell JW;Udhayakumar V
分子诊断方法可以补充现有的工具，以改善疟疾的诊断。然而，它们需要良好的实验室基础设施，从而限制了它们仅用于参考实验室和研究。因此，在疟疾流行国家采用常规使用的分子工具将需要更简单的分子平台。最近开发的环介导等温扩增（LAMP）方法相对简单，可以改进，以便更好地用于流行国家。在这项研究中，我们试图通过使用能够在一个平台中进行LAMP的扩增和检测（通过荧光）的简单便携式装置来改进这种用于疟疾诊断的方法。我们称之为Realtime方法。利用已发表的属特异性引物对该方法进行了验证。来自不同种类的疟疾寄生虫的DNA用于初始表征。恶性疟原虫的临床样本被用来确定该系统的灵敏度和特异性相比，显微镜和巢式PCR方法。此外，直接煮沸的寄生虫制剂进行了比较，与传统的DNA分离方法。RealPCR方法被认为是简单的，并允许实时检测DNA扩增。扩增时间不同，但通常小于60分钟。检测到所有感染人类的疟原虫。与镜检法相比，Realcidin检测恶性疟原虫的敏感性和特异性分别为96.7%和91.7%，与标准巢式PCR法相比，Realcidin检测恶性疟原虫的敏感性和特异性分别为98.9%和100%。此外，该方法一致地从直接煮沸的血液样品中检测出恶性疟原虫。这种Realtime方法具有很大的潜力，作为一个现场可用的分子工具，用于诊断疟疾。该工具可以为临床和操作程序中的现场使用提供传统的基于PCR的诊断方法的替代方案。
Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.
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