Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection.
Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection.
复制标题
可视化环介导等温扩增技术在间日疟原虫感染现场检测中的应用
DOI：
10.1186/1756-3305-4-115
复制
发表时间：
2011-06-21
影响因子：
3.2
通讯作者：
Gao Q
中科院分区：
文献类型：
--
作者：
Tao ZY;Zhou HY;Xia H;Xu S;Zhu HW;Culleton RL;Han ET;Lu F;Fang Q;Gu YP;Liu YB;Zhu GD;Wang WM;Li JL;Cao J;Gao Q
背景
环介导等温扩增（LAMP）是一种高效的DNA检测方法，有望用于感染性病原体（包括疟原虫属）的分子检测。然而，在大多数疟疾流行地区，这往往是有限的资源，目前的LAMP方法是不可行的诊断，由于准确地解释结果的困难与问题的敏感可视化的扩增产物，和污染的风险导致的大量扩增的DNA产生。在这项研究中，我们建立了一个新的可视化LAMP方法在封闭的管道系统，并验证它的疟疾诊断模拟现场条件下。
方法
通过在反应开始之前将含有高灵敏度DNA荧光染料SYBR绿色I的微晶蜡染料胶囊添加到正常的LAMP反应中来建立可视化的LAMP方法。共收集89份血样，滤纸收集，并使用简单的煮沸法处理的DNA提取，然后用可视化的LAMP方法检测间日疟原虫感染。
结果
蜡囊在等温扩增期间保持完整，并且仅当扩增后温度升高至熔点时才将DNA染料释放至反应混合物。冷却后不久，固化的蜡将反应混合物密封在管的底部，从而最大限度地降低了气溶胶污染的风险。与镜检法相比，LAMP的敏感性和特异性分别为98.3%（95%置信区间（CI）：91.1-99.7%）和100%（95% CI：88.3-100%），并与巢式聚合酶链反应方法密切一致。
结论
这种新颖、廉价、快速的可视化LAMP方法在资源有限的现场环境中用于疟疾诊断是可行的。
Background
Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions.
Methods
A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection.
Results
The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method.
Conclusions
This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.
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影响因子：
3.5
作者：
Long, Earl G.
通讯作者：
Long, Earl G.
影响因子：
9.4
作者：
Parida, M;Posadas, G;Morita, K
通讯作者：
Morita, K
影响因子：
3.2
作者：
Aonuma H;Yoshimura A;Perera N;Shinzawa N;Bando H;Oshiro S;Nelson B;Fukumoto S;Kanuka H
通讯作者：
Kanuka H
影响因子：
9.4
作者：
Polley, Spencer D.;Mori, Yasuyoshi;Sutherland, Colin J.
通讯作者：
Sutherland, Colin J.
影响因子：
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作者：
Mori, Y;Kitao, M;Notomi, T
通讯作者：
Notomi, T