A novel dengue virus detection method that couples DNAzyme and gold nanoparticle approaches.
A novel dengue virus detection method that couples DNAzyme and gold nanoparticle approaches.
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DOI：
10.1186/1743-422x-10-201
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发表时间：
2013-06-28
期刊：
影响因子：
4.8
通讯作者：
Fraser MJ Jr
中科院分区：
文献类型：
--
作者：
Carter JR;Balaraman V;Kucharski CA;Fraser TS;Fraser MJ Jr
最近登革热病毒（DENV）的流行加上即将出现的新的爆发，重新需要新的检测方法，该方法能够在症状表现之前识别这种病毒病原体。及时检测DENV的能力对于从疾病症状中快速恢复至关重要。一种修饰的实验室衍生的10-23 DNAzyme与金纳米颗粒相连，为检测病毒（如DENV）提供了强大的工具。我们研究了将DNAzyme（DDZ）活化与盐诱导的金纳米颗粒（AuNP）聚集偶联以检测蚊子细胞中的登革病毒（DENV）后代的有效性。设计DNA酶以识别在所有DENV中保守的5'环化序列（5' CS），并与AuNP缀合。DDZ-AuNP已经证明能够检测从感染的白纹伊蚊C6/36细胞中分离的我们的模型登革热株DENV-2 NGC的基因组RNA。这些靶向事件导致AuNP的快速聚集，导致反应混合物的红色到透明颜色的转变，从而导致DENV RNA基因组的阳性检测。在反应混合物中包含SDS允许直接从细胞培养上清液检测DENV，而无需额外的样品处理。特异性试验证明检测是DENV特异性的，而灵敏度试验证实在1 × 101 TCID 50单位水平下检测。这些结果表明DDZ-AuNP以序列特异性方式和在现场使用的实际浓度下有效地检测DENV基因组。我们已经开发了一种有效的检测试验，使用DNAzyme催化与AuNP聚集偶联，用于以序列特异性方式检测DENV基因组。我们的新型DDZ-AuNP检测方法的全面发展将为在几分钟内检测蚊子细胞和组织中的DENV以及可能感染的患者血清提供实用，快速和低成本的替代方案，几乎不需要专门的培训。
Recent epidemics of dengue viruses (DENV) coupled with new outbreaks on the horizon have renewed the demand for novel detection methods that have the ability to identify this viral pathogen prior to the manifestation of symptoms. The ability to detect DENV in a timely manner is essential for rapid recovery from disease symptoms. A modified lab-derived 10-23 DNAzyme tethered to gold nanoparticles provides a powerful tool for the detection of viruses, such as DENV. We examined the effectiveness of coupling DNAzyme (DDZ) activation to the salt-induced aggregation of gold nanoparticles (AuNP) to detect dengue virus (DENV) progeny in mosquito cells. A DNAzyme was designed to recognize the 5’ cyclization sequence (5’ CS) that is conserved among all DENV, and conjugated to AuNPs. DDZ-AuNP has demonstrated the ability to detect the genomic RNA of our model dengue strain, DENV-2 NGC, isolated from infected Aedes albopictus C6/36 cells. These targeting events lead to the rapid aggregation of AuNPs, resulting in a red to clear color transition of the reaction mixes, and thus positive detection of the DENV RNA genome. The inclusion of SDS in the reaction mixture permitted the detection of DENV directly from cell culture supernatants without additional sample processing. Specificity assays demonstrated detection is DENV-specific, while sensitivity assays confirm detection at levels of 1 × 101 TCID50 units. These results demonstrate DDZ-AuNP effectively detects DENV genomes in a sequence specific manner and at concentrations that are practical for field use. We have developed an effective detection assay using DNAzyme catalysis coupled with AuNP aggregation for the detection of DENV genomes in a sequence specific manner. Full development of our novel DDZ-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of DENV in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required.