Selective Determination of Isothermally Amplified Zika Virus RNA Using a Universal DNA-Hairpin Probe in Less than 1 Hour

Abstract

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/μL (∼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.

MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures

“…The design of the MVF sensor was based on the 4WJ sensor design reported by us earlier . To improve the sensor's ability to interrogate highly structured RNA targets, we incorporated strand V in addition to strands M and F in the sensor design (Scheme ).…”

Detection of RNA viruses from influenza and HIV to Ebola and SARS-CoV-2: a review

“…Results can be obtained rapidly (within an hour), which makes these tests a suitable platform to be used for the development of point-of-care diagnostics tests. Alongside LAMP, the NASBAbased nucleic acid detection, 46,55 as well as the advanced strand exchange amplication-based nucleic acid detection, are used and demonstrate a low limit of detection. A standard PCR test can also be incorporated into the assay, with other methods of separation such as laser-irradiated DNA extraction, paramagnetic particle separation, and others.…”

“…Electrochemical detection methods provide sensitive detection of a wide range of analytes: virions, 40 viral antigen, 44 antibodies to the virus, 33 and viral nucleic acids. 46 The main advantage of these detection methods is that they are relatively inexpensive and not limited by a diffraction limit (like optical methods). The lowest limit of detection was reported by Teeparuksapun et al 28 to be at a subattogram per milliliter concentration.…”

A Label-Free Impedimetric Genosensor for the Nucleic Acid Amplification-Free Detection of Extracted RNA of Dengue Virus

“…Experimentally, specific sequences used as probe DNA (pDNA) are immobilized on the surface of various electrodes, and then the electrochemical signal, after hybridization, is directly quantified by electrocatalytic electrodes, such as Cu 2 CdSnS 4 /O 2 /Si [ 17 ], graphite electrode [ 18 ] and indium tin oxide (ITO) [ 26 ], and evaluated by using electroactive intercalator, such as methylene blue [ 19 , 27 ], and mediators, such as ferrocyanide/ferricyanide [ 15 , 16 , 20 , 28 ]. While most publications on electrochemical DNA genosensors used synthetic DNA (≤200-mer) or nucleic acid amplification RNA fragments (<150-mer) as the target analytes to elucidate the sensing performance of genosensors [ 15 , 17 , 18 , 19 , 20 , 27 , 28 ], only one study detected the extracted RNA fragments of DENV [ 16 ]. Mills et al [ 27 ] and Lynch III et al [ 28 ] developed four-way junction-based genosensors to solve the influence of overhangs on the detection of synthetic DNA (200-mer) and Zika RNA amplicon (147-mer), respectively.…”

“…While most publications on electrochemical DNA genosensors used synthetic DNA (≤200-mer) or nucleic acid amplification RNA fragments (<150-mer) as the target analytes to elucidate the sensing performance of genosensors [ 15 , 17 , 18 , 19 , 20 , 27 , 28 ], only one study detected the extracted RNA fragments of DENV [ 16 ]. Mills et al [ 27 ] and Lynch III et al [ 28 ] developed four-way junction-based genosensors to solve the influence of overhangs on the detection of synthetic DNA (200-mer) and Zika RNA amplicon (147-mer), respectively. Presently, there is little research exploring the nucleic acid amplification-free detection by a genosensor, because it is difficult to control the total length of directly extracted targets with length-varied overhangs, which may hinder the hybridization efficiency.…”