Integrated microsystems for the in situ genetic detection of dengue virus in whole blood using direct sample preparation and isothermal amplification

摘要

Owing to the frequent outbreak of dengue fever worldwide, a highly sensitive but in situ simple process diagnostic device is required to detect the dengue virus. However, the current immune affinity-based methods have sensitivity issues and nucleic acid-based diagnostic devices have not been suitable for field diagnosis due to the complexity in sample preparation. Here, a simple and fast nucleic acid-based diagnostic tool to directly detect dengue viruses in whole blood is demonstrated using a microbead-assisted direct sample preparation buffer (MB-buffer) and isothermal amplification (loop-mediated isothermal amplification, LAMP). To maximize the performance of the sample preparation process in the microfluidic chip platform, the chemical composition of the sample preparation buffer is simplified and combined with physical tools (heating and bead beating). The entire serial processes consisted of only (1) sample (whole blood) loading, (2) stirring for 90 s, (3) heating at 70 °C for 10 min, and (4) LAMP amplification in the simply designed microfluidic chip cartridge. A single syringe was utilized for sample loading and microfluidic solution transfer. Consequently, dengue viruses were qualitatively detected and discriminated with high sensitivity (LOD: 102 PFU per 200 μL of whole blood) in less than 1 hour without the use of any sophisticated system.