Public Health Virology Laboratory, Forensic and Scientific Services, Queensland Health, Brisbane, Queensland, Australia.
Metro South Public Health Unit, Metro South Hospital and Health Service, Brisbane, Queensland, Australia.
PLoS Negl Trop Dis. 2020 Mar 4;14(3):e0008130. doi: 10.1371/journal.pntd.0008130. eCollection 2020 Mar.
Yellow fever, dengue, chikungunya and Zika viruses are responsible for considerable morbidity and mortality in humans. Aedes aegypti and Aedes albopictus are the most important mosquito vectors involved in their transmission. Accurate identification of these species is essential for the implementation of control programs to limit arbovirus transmission, during suspected detections at ports of first entry, to delimit incursions or during presence/absence surveillance programs in regions vulnerable to invasion. We developed and evaluated simple and rapid colorimetric isothermal tests to detect these two mosquito species based on loop-mediated isothermal amplification (LAMP) targeting the ribosomal RNA internal transcribed spacer 1 (ITS1).
METHODOLOGY/PRINCIPAL FINDINGS: Samples were prepared by homogenizing and heating at 99 oC for 10 min before an aliquot was added to the LAMP reaction. After 40 min incubation at 65 oC, a colour change indicated a positive result. The tests were 100% sensitive and species-specific, and demonstrated a limit of detection comparable with PCR-based detection (TaqMan chemistry). The LAMP assays were able to detect target species for various life stages tested (adult, 1st instar larva, 4th instar larva and pupa), and body components, such as legs, wings and pupal exuviae. Importantly, the LAMP assays could detect Ae. aegypti DNA in mosquitoes stored in Biogents Sentinel traps deployed in the field for 14 d. A single 1st instar Ae. aegypti larva could also be detected in a pool of 1,000 non-target 1st instar Aedes notoscriptus, thus expediting processing of ovitrap collections obtained during presence/absence surveys. A simple syringe-sponge protocol facilitated the concentration and collection of larvae from the ovitrap water post-hatch.
CONCLUSIONS/SIGNIFICANCE: We describe the development of LAMP assays for species identification and demonstrate their direct application for surveillance in different field contexts. The LAMP assays described herein are useful adjuncts to laboratory diagnostic testing or could be employed as standalone tests. Their speed, ease-of-use, low cost and need for minimal equipment and training make the LAMP assays ideal for adoption in low-resource settings without the need to access diagnostic laboratory services.
黄热病、登革热、基孔肯雅热和寨卡病毒可导致人类出现大量发病和死亡。埃及伊蚊和白纹伊蚊是与这些病毒传播有关的最重要的蚊媒。准确识别这些物种对于实施控制计划至关重要，这些计划旨在限制虫媒病毒的传播，包括在港口首次入境时对疑似病例的检测、划定入侵范围，以及在易受入侵地区进行存在/不存在监测计划时。我们开发并评估了基于核糖体 RNA 内部转录间隔区 1（ITS1）的环介导等温扩增（LAMP）的简单快速比色等温检测法，以检测这两种蚊子。
方法/主要发现：在将样品加入 LAMP 反应之前，将其匀浆并在 99°C 下加热 10 分钟。在 65°C 孵育 40 分钟后，颜色变化表示阳性结果。这些测试的灵敏度为 100%，且具有物种特异性，检测限与基于 PCR 的检测（TaqMan 化学法）相当。LAMP 检测法能够检测到各种测试的生命阶段的目标物种（成虫、1 龄幼虫、4 龄幼虫和蛹），以及身体部位，如腿、翅膀和蛹蜕。重要的是，LAMP 检测法可以检测到在野外使用 Biogents Sentinel 诱捕器放置 14 天后捕获的埃及伊蚊 DNA。即使在含有 1000 只非目标白纹伊蚊 1 龄幼虫的样本中，也能检测到单个埃及伊蚊 1 龄幼虫，从而加快了在有无监测期间对卵胎生收集器的处理。简单的注射器-海绵方案便于从卵胎生收集器的水中收集孵化后的幼虫。
结论/意义：我们描述了用于物种鉴定的 LAMP 检测法的开发，并证明了它们在不同野外环境中的直接应用。本文所述的 LAMP 检测法可作为实验室诊断检测的辅助手段，也可作为独立检测方法。它们的速度快、易于使用、成本低，且仅需最低限度的设备和培训，非常适合在资源匮乏的环境中采用，而无需获得诊断实验室服务。
