Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone.
Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone.
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DOI：
10.1371/journal.pntd.0006671
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发表时间：
2018-08-01
影响因子：
3.8
通讯作者：
Ellington, Andrew D
中科院分区：
文献类型：
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作者：
Bhadra, Sanchita;Riedel, Timothy E;Ellington, Andrew D
使用内共生细菌Wolbachia操纵天然蚊子种群正在研究作为一种新的策略，以减少蚊子传播的病毒的负担。为了评估这些干预措施的有效性，确定埃及伊蚊种群中的沃尔巴克氏体感染频率至关重要。然而，目前的诊断工具并不能很好地满足这一需求。形态学方法无法识别沃尔巴克氏体，免疫测定通常灵敏度低，通量差，而PCR和光谱学需要复杂的仪器和技术专长，这限制了它们在集中实验室的使用。为了解决这一未满足的需求，我们使用环介导等温扩增（LAMP）和寡核苷酸链置换（OSD）探针来创建用于载体和共生体监测的一锅法样品到答案核酸诊断平台。LAMP-OSD测定可以直接扩增来自浸软的蚊子的靶核酸，而不需要核酸纯化，并产生眼睛或手机相机可观察到的特异性单终点是/否荧光信号。我们展示了两个目标，埃及伊蚊细胞色素氧化酶I（coi）基因和沃尔巴克氏体表面蛋白（wsp）基因的手机成像LAMP-OSD测试，并显示4和40个目标DNA拷贝的检测限，分别。在对90只现场捕获的蚊虫进行盲法试验中，COI LAMP-OSD检测法对Ae的鉴定显示出98%的特异性和97%的灵敏度。即使在37°C下不使用干燥剂储存3周后，也不会对埃及伊蚊产生任何影响。类似地，wsp LAMP-OSD测定容易地鉴定现场收集的白纹伊蚊蚊子中的wAlbB沃尔巴克氏体菌株，而不产生任何假阳性信号。适度的技术要求，最少的执行步骤，简单的二进制读出，和强大的准确性，使LAMP-OSD手机检测平台非常适合在严峻或资源有限的条件下进行现场矢量监测。
Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and technical expertise, which restrict their use to centralized laboratories. To address this unmet need, we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids from macerated mosquitoes without requiring nucleic acid purification and yield specific single endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti mosquitoes even after 3 weeks of storage without desiccant at 37°C. Similarly, the wsp LAMP-OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus mosquitoes without generating any false positive signals. Modest technology requirements, minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-cellphone assay platform well suited for field vector surveillance in austere or resource-limited conditions.