Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP).
Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP).
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DOI：
10.1371/journal.pone.0169011
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Carlow CK
中科院分区：
文献类型：
--
作者：
Poole CB;Li Z;Alhassan A;Guelig D;Diesburg S;Tanner NA;Zhang Y;Evans TC Jr;LaBarre P;Wanji S;Burton RA;Carlow CK
准确检测人体丝虫寄生虫对于控制盘尾丝虫病和淋巴丝虫病的大规模药物管理计划的实施和评估至关重要。确定媒介群体的感染水平对于评估传播、决定何时终止药物治疗以及监测复发也很重要。可以使用免疫学方法来检测人类感染，但是，已经报道了交叉反应问题。基于核酸的分子检测具有高水平的特异性和敏感性，可用于检测人类和载体的感染。在这项研究中，我们开发了环介导等温扩增 (LAMP) 测试，使用 pH 敏感染料来检测人类和昆虫样本中的三种不同丝虫 DNA，以增强扩增的视觉检测。此外，反应在便携式非仪器核酸扩增 (NINA) 装置中进行，该装置为 LAMP 提供稳定的热源。结合中性红或酚红染料评估了几种链置换 DNA 聚合酶的功效。比色 NINA-LAMP 检测针对丝虫 Hha I 重复序列、盘尾丝虫 GST1a 和班氏吴切菌 LDR，均表现出物种特异性，并且灵敏度很高，检测的 DNA 相当于一种微丝蚴的 1/10-1/5000。反应时间根据是否采用单拷贝基因（70 分钟，盘旋丝虫）或重复 DNA（40 分钟，马来芽孢杆菌和班克罗夫蒂丝虫）作为生物标志物而变化。 NINA 加热器可用于同时检测多种感染。该技术的准确性、简单性和多功能性表明比色 NINA-LAMP 检测非常适合监测丝虫病控制计划的成功情况。
Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.