Firebird Biomolecular Sciences LLC, 13709 Progress Blvd, Box 17, Alachua, FL, 32615, USA.
Florida Medical Entomology Laboratory, University of Florida, 200 9th Street SE, Vero Beach, FL, 32962, USA.
BMC Infect Dis. 2019 May 14;19(1):418. doi: 10.1186/s12879-019-3998-z.
The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed.
We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides).
The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3-4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels.
Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.
登革热（DENV）、基孔肯雅热（CHIKV）和寨卡病毒（ZIKV）在全球范围内的传播对公共卫生造成了严重影响。由于这些虫媒病毒由同一蚊种传播，并且在同一地区共同循环，因此需要一种能够同时检测它们、具有区分能力的灵敏诊断检测方法。
我们在此展示了一种基于病毒 RNA 的逆转录-PCR 扩增的诊断试剂盒，以及一种涉及 PCR 扩增子和病毒特异性探针直接杂交的 xMap Luminex 架构。两种 DNA 创新（“人为扩展遗传信息系统”（AEGIS）和“自回避分子识别系统”（SAMRS））与标准方法（标准核苷酸）相比，提高了 Luminex 微球上的杂交灵敏度和多重检测的 PCR 特异性。
该诊断试剂盒可检测到这些病毒，如果存在的话，其分辨率为 20 个基因组当量（DENV1），或 10 个（DENV3-4、CHIKV）和 80 个（DENV2、ZIKV）基因组当量/检测。它可在单个感染的蚊子中、由 5 到 50 只个体组成的蚊子池中以及蚊子唾液（ZIKV、CHIKV 和 DENV2）中识别 ZIKV、CHIKV 和 DENV RNA。感染的蚊子和唾液也被收集在阳离子表面（Q-纸）上，该表面通过静电作用结合蚊子和病毒的核酸。来自感染蚊子的所有样本仅显示出靶标特异性信号；来自非感染样本的信号处于背景水平。
我们的结果提供了一种高效的多重工具，可用于监测新出现的蚊媒病原体，有助于在高传播风险地区进行有针对性的蚊子控制。
