Sequence-specific detection of different strains LCMV in a single sample using tentacle probes

Abstract Viral infections often result in quasi-species of virus strains that can have dramatic impacts on disease outcomes or experimental results. 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 in order to design appropriate treatment, and can be useful for many experimental systems. We have developed a novel molecular method for simple and accurate detection of RNA virus genetic variants using Tentacle probes coupled with quantitative PCR. Tentacle probes are modified molecular beacons that are less expensive and dramatically improve false positive rates in routine qPCR. To validate this approach we have designed two tentacle probes for two strains of Lymphocytic Choriomeningitis Virus (LCMV): the parental Armstrong strain and the more virulent clone-13 strain, which differ by only 3 nucleotide substitutions. Using this approach we were able to distinguish these two strains of LCMV. Furthermore, in virus mixing experiments we were able to detect down to 5% of clone-13 diluted in Armstrong, and vice versa. Thus, we have developed a fast, cost-effective approach for differentiating between closely related viruses.

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