A nucleic acid-based lateral flow device for rapid diagnosis of West Nile Virus infection

When epidemic outbreaks occur, fast screening of humans and/or animals as well as environmental monitoring of pathogens in the respective region is essential to locally contain the spread of the disease and prevent pandemics. Rapid diagnostic tests are a valuable tool in this context. Within our project MOBILISE, which is dedicated to establishing a mobile laboratory facility responding to disease outbreaks in remote areas, we are developing nucleic acid-based lateral flow devices (LFDs) as rapid tests for West Nile Virus (WNV). WNV has become more common in Europe in recent years due to increasing temperatures caused by climate change [1]. Nucleic acid-based LFDs directly target viral nucleic acids using DNA probes suitable for hybridization to the target sequence. Our approach involves immobilization of oligonucleotides complementary to a segment of the target WNV sequence on the test line as a capture probe. A gold nanoparticle-DNA-horseradish peroxidase conjugate containing oligonucleotides that bind a different section of the WNV target nucleic acid serves as detection probe to visualize the signal, with Au nanoparticles accumulating on the test line. The oligonucleotides immobilized on the gold nanoparticle and test line are derived from the forward primer and the reverse complement of the reverse primer of a published WNV PCR assay. In the rapid test developed herein, the optical signal is further enhanced by applying 3,3′,5,5′-tetramethylbenzidin, which is converted to a blue product by the horseradish peroxidase immobilized on the Au-nanoparticle-DNA-HRP conjugate. We varied the ratio of DNA loaded onto the Au nanoparticles to optimize sensitivity of our lateral flow device, which was lowest (1 nM WNV ssDNA) at a ratio of 63:1 of DNA:AuNP and 0.25 mg/ml HRP during conjugate synthesis. In conclusion, we have developed a nucleic-acid-based lateral flow device for WNV detection which directly detects viral nucleic acid without using antibodies. Targeting viral nucleic acids is beneficial for identifying pre-symptomatic and acute cases, where antibody titres are zero or low [2]. Furthermore, a nucleic-acid-based approach avoids frequently encountered problems of antibody-based rapid tests such as batch-to batch variations in antibody quality and enables rapid adaptation to new virus variants by simply changing the sequences of capture, detection and control probes. Future work will focus on improving sensitivity by optimizing the composition of the running buffer and implementing pre-amplification of the target into the LFD. 

References 

1. Farooq, Z. et al., 2023. One Health 16: 100509. 

2. Lustig, Y. et al., 2018. Frontiers in Microbiology 9: 2421.