Selective Determination of Isothermally Amplified Zika Virus RNA Using a Universal DNA-Hairpin Probe in Less than 1 Hour
Selective Determination of Isothermally Amplified Zika Virus RNA Using a Universal DNA-Hairpin Probe in Less than 1 Hour
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DOI：
10.1021/acs.analchem.9b02455
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发表时间：
2019-11-05
影响因子：
7.4
通讯作者：
Chumbimuni-Torres, Karin Y.
中科院分区：
文献类型：
--
作者：
Lynch, Charles A., III;Foguel, Marcos V.;Chumbimuni-Torres, Karin Y.
最近在美洲爆发的寨卡病毒（ZIKV）以及将该病毒与小头畸形和神经系统并发症病例联系起来的多项研究表明，需要具有成本效益和快速的ZIKV诊断测试。在此，开发了一种诊断平台，其依赖于基于四向结（4 WJ）的生物传感器，其具有使用通用DNA-发夹（UDH）探针的电化学读出，用于等温扩增的ZIKV RNA片段的选择性识别。4 WJ结构利用电极固定的茎-环（DNA-发夹）探针和与茎-环探针和ZIKV扩增子的靶向片段互补的两条DNA衔接子链。其中一条衔接子链负责靶识别的高选择性，而另一条衔接子链有助于解旋靶二级结构。第一个衔接子链包含氧化还原标记亚甲基蓝，以响应电极表面上4 WJ结构的靶依赖性形成而触发电流变化。扩增子可以直接从扩增样品中分析而不需要纯化。所提出的诊断方法表现出1.11 fg/mu L（类似于0.3 fM）的ZIKV RNA检测极限和允许可靠区分ZIKV与西尼罗河病毒和四种登革热病毒血清型的高选择性。总体而言，ZIKV RNA的分析可以在不到1小时内完成，包括扩增和电化学检测。
The recent outbreak of the Zika virus (ZIKV) in the Americas and multiple studies that linked the virus to the cases of microcephaly and neurological complications have revealed the need for cost efficient and rapid ZIKV diagnostics tests. Here, a diagnostic platform relying on a four-way junction (4WJ)-based biosensor with electrochemical readout using a Universal DNA-Hairpin (UDH) probe for the selective recognition of an isothermally amplified ZIKV RNA fragment is developed. The 4WJ structure utilizes an electrode-immobilized stem-loop (DNA-hairpin) probe and two DNA adaptor strands complementary to both the stem-loop probe and the targeted fragment of a ZIKV amplicon. One of the adaptor strands is responsible for high selectivity of the target recognition, while another helps unwinding the target secondary structure. The first adaptor strand contains a redox label methylene blue to trigger the current change in response to the target-dependent formation of the 4WJ structure on the surface of the electrode. The amplicon can be analyzed directly from the amplification sample without the need for its purification. The proposed diagnostic methodology exhibits the limit of ZIKV RNA detection of 1.11 fg/mu L (similar to 0.3 fM) and high selectivity that allows for reliable discrimination of ZIKV from West Nile virus and four dengue virus serotypes. Overall, the analysis of ZIKV RNA can be completed in less than 1 h, including amplification and electrochemical detection.