Background: Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems.

Methods: We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe.

Results: Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA.

Conclusions: Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.

Antibody detection and accurate diagnosis of tropical diseases is essential to help prevent the spread of disease. However, most detection methods lack cost-effectiveness and field portability, which are essential features for achieving diagnosis in a timely manner. To address this, 3D-printed oblate spheroid sample chambers were fabricated to measure green light scattering of gold nanoparticles using an optical caustic focus to detect antibodies. Scattering signals of 20–200 nm gold nanoparticles using a green laser were compared to green light emitting diode (LED) light source signals and to Mie theory. The change in signal from 60 to 120 nm decreased in the order of Mie Theory > optical caustic scattering > 90° scattering. These results suggested that conjugating 60 nm gold nanoparticles and using an optical caustic system to detect plasmonic light scattering, would result in a sensitive test for detecting human antibodies in serum. Therefore, we studied the light scattering response of conjugated gold nanoparticles exposed to different concentrations of anti-protein E antibody, and a feasibility study of 10 human serum samples using dot blot and a handheld optical caustic-based sensor device. The overall agreement between detection methods suggests that the new sensor concept shows promise to detect gold nanoparticle aggregation in a homogeneous assay. Further testing and protocol optimization is needed to draw conclusions on the positive and negative predictive values for this new testing system.