Enzyme-free amplification and detection of bovine viral diarrhea virus RNA using hybridization chain reaction and gold nanoparticles

Abstract

A novel bovine viral diarrheal virus (BVDV)-RNA detection method was developed using a combination of the amplification capability of hybridization chain reaction (HCR) with the sensitivity of an unmodified-gold nanoparticle (AuNP) colorimetric detection assay. Two auxiliary probes were designed to target a conserved RNA sequence among the BVDV isolates. The complementary target BVDV-RNA was used as the initiator to trigger a cascade of hybridization events to yield nicked double-helix DNA analogous to the alternating copolymers. DNA in the form of a nicked double helix did not prevent salt-induced aggregation of AuNPs. In contrast, in the absence of the complementary target BVDV-RNA, free hairpins with single-stranded sticky ends adsorbed onto the AuNPs, stabilize them, and prevent salt-induced aggregation of the AuNP. The limit of detection (LOD) for the BVDV-RNA was estimated to be 0.008 tissue culture infective dose (TCID₅₀)/reaction. The method developed was highly selective and specific to detect BVDV isolates in clinical samples. This protocol offers a rapid, simple, and cost-effective assay for detecting BVDV.

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