Development and Comparison of Cross-Linking and Non-Crosslinking Probe-Gold Nanoparticle Hybridization Assays for Direct Detection of Unamplified Bovine Viral Diarrhea Virus-RNA

Abstract

Background: Bovine viral diarrhea virus (BVDV) is a major economic disease that has been spread in most countries. In addition to vaccination, one of the main ways to control the disease and prevent it from spreading is to detect and cull infected animals, especially those with persistent infection (PI). We developed and compared two colorimetric biosensor assays based on probe-modified gold nanoparticles (AuNPs) to detect BVDV. Specific probes were designed to detect the 5ʹ untranslated region of BVDV-RNA. The thiolated probes were immobilized on the surface of the AuNPs. Two methods of cross-linking (CL) and non-crosslinking (NCL) probe-AuNPs hybridization were developed and compared. Results: The hybridization of positive targets with the two probe-AuNPs formed a polymeric network between the AuNPs which led to the aggregation of nanoparticles and color change from red to blue. Alternatively, in the NCL mode, the hybridization of complementary targets with the probe-AuNPs resulted in the increased electrostatic repulsion in nanoparticles and the increased stabilization against salt-induced aggregation. The CL and NCL assays had detection limits of 6.83 and 44.36 ng/reaction, respectively. Conclusion: The CL assay showed a higher sensitivity and specificity; in contrast, the NCL assay did not require optimizing and controlling of hybridization temperature and showed a higher response speed. However, both the developed methods are cost-effective and easy to perform and also could be implemented on-site or in local laboratories in low-resource countries.