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 5′ 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.

Diagnosis of bovine viral diarrhea virus: an overview of currently available methods

“…The hybridization of positive targets with the probe-AuNPs resulted in the formation of a polymeric network among AuNPs, ultimately leading to the aggregation of nanoparticles and color change from red to blue. The cross-linking (CL) and non-crosslinking (NCL) probe-AuNPs assays demonstrated detection limits of 6.83 and 44.36 ng/reaction, respectively (Heidari et al, 2021). Kim et al developed an electrochemical biosensor system for the rapid BVDV detection using synthetic conductive nanomaterials black phosphorus (BP) and AuNPs.…”

Non-structural proteins of bovine viral diarrhea virus

“…To date, most vaccines have been developed by inactivated pathogens, protein subunits, or based on live attenuated organisms, which are at risk of pathogenicity recovery under the condition of the impaired immune system. To avoid this situation, the research and development of nanoparticles-based vaccines that enhance the stability and targeting of antigens have attracted attention [ 90 – 92 ]. Mahony et al combined E2 protein with hollow mesoporous silica nanoparticles with surface amino functionalization (HMSA) to make a recombinant subunit vaccine, and immunized sheep by subcutaneous injection of non-lyophilized or freeze-dried nano-formulations.…”

Design of an optical nanobiosensor for detection of Legionella pneumophila in water samples

“…It uses two nucleic acid nanoprobes (Np-DNA), each of which complements a portion of the target gene. These nanoprobes hybridize in the presence of the target gene and form a polymeric network of nanoparticles and nucleic acid as they approach each other, resulting in a change in the color of the solution from red to purple ( 21 , 22 ). In the Non-Cross-Linking (NCL), one (Np-DNA) is used, which alone complements the entire identified gene sequence.…”