A Novel Unmodified Gold Nanoparticles-Based Assay for Direct Detection of Unamplified Bovine Viral Diarrhea Virus-RNA

摘要

Bovine viral diarrhea (BVD)is one of the most prevalent infectious diseases of bovines in worldwide and is responsible for severe economic losses in livestock production. A simple method based on unmodified gold nanoparticles (AuNPs) was developed for the detection of BVD virus-1 and -2. The different properties of AuNPs in the presence of single-and double-stranded oligonucleotides were exploited for developing a hybridization assay for the direct detection of BVDV-RNA. The assay involved hybridization of the specific probes and target RNA, followed by the addition of AuNPs to suspensions. Aggregation of AuNPs in the presence of dsDNA-RNA hybrid in a salt-containing buffer resulted in color change of the solution from red to blue, whereas in the absence of target RNA, probes were free to stabilize AuNPs, and the suspension color remained red. The assay allows the detection of RNA by naked eye from as low as two hundred 50% tissue culture infective doses (TCID50) of BVDV per mL. The diagnostic sensitivity and specificity of this method accordance with multiplex nested RT-PCR were found to be 90% and 100% respectively. The assay using unmodified AuNPs provides the detection of BVDV-RNA in a label-and enzyme-free manner through visual observation of color change.