Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.
Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.
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DOI：
10.1016/j.actatropica.2016.06.009
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发表时间：
2016-10
期刊：
影响因子：
2.7
通讯作者：
Warburg A
中科院分区：
文献类型：
--
作者：
Abbasi I;Kirstein OD;Hailu A;Warburg A
建立了3种用于利什曼病诊断的环介导等温扩增(LAMP)系统。绿色核酸染色Syto-16用于实时监测反应。LAMP法检测100Fg DNA/反应具有很高的灵敏度。在流行地区的大量无症状个体中检测到利什曼原虫DNA。内脏利什曼病(VL)是由杜氏利什曼原虫属原虫引起的一种重要的被忽视的热带疾病，主要流行于印度次大陆、东非和巴西。通过聚合酶链式反应扩增ITS1和/或kDNA基因可诊断VL。目前的研究包括优化环路介导的等温扩增(LAMP)检测人类血液或组织样本中的利什曼原虫DNA。建立了三个LAMP系统；其中两个是根据不同利什曼原虫ITS1基因的共同区域设计的，而第三个LAMP系统的引物是根据利什曼原虫基因组中新发现的重复区域设计的。LAMP测试被证明足够灵敏，可以从大多数利什曼原虫物种中检测出0.1万亿pg的DNA。首次使用绿色核酸染色SYTO16，以实现对LAMP扩增的实时监测。讨论了用Syto 16实现实时灯检测相对于终端灯产品检测的优势。比较实时荧光检测方法与qRT-kDNA聚合酶链式反应(qRT-kDNA)检测利什曼原虫DNA的效果。
Three systems of loop-mediated isothermal amplification (LAMP) were developed for diagnosing leishmaniasis. The green nucleic acid stain, SYTO-16 was adapted for monitoring the reactions in real-time. The LAMP assays proved highly sensitive detecting >100Fg DNA/reaction. Leishmania DNA was detected in a significant number of asymptomatic individuals living in endemic areas. Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1 pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR.