A Methodology for Porcine Circovirus 2 (PCV-2) Quantification Based on Gold Nanoparticles

Abstract

The aim of the current study is to introduce a methodology aimed at producing a biosensor that uses gold nanoparticles (AuNPs) to detect porcine circovirus 2 (PCV-2). This biosensor was based on AuNPs, which were modified with self-assembled monolayers (SAMs) and antibodies. The AuNPs' surface and virus modification process applied to enable antibody binding was accompanied by localized surface plasmon resonance (LSPR), surface plasmon resonance (SPR), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDX). Virus quantification was possible by the light absorption difference in the spectrum at concentrations of 10 5 , 10 6 , 10 7 , 10 8 , and 10 9 DNA copies/mL PCV-2 in relation to quantitative PCR (qPCR), with an R 2 value >0.98. The visualization of colorimetric changes in the different PCV-2 concentrations was possible without the use of equipment. The biosensor production methodology presented reproducibility and specificity, as well as easy synthesis and low cost. An enhanced version of it may be used in the future to replace traditional tests such as PCR.

Colorimetric Kit for Rapid Porcine Circovirus 2 (PCV-2) Diagnosis

“…PCV-2 antibodies were added to the solution after modifications were carried out on the surface of hybrid nanoparticles to enable covalent binding between antibody amines and nanoparticles. The herein-used antibodies concentration was 2.5 µg/mL (30 µL) based on previous study by our group; the color of the solution remained purple [ 14 ]. The first part of the analyses was carried out to find the best dilution to be adopted.…”

“…Moreover, qPCRs were performed based on using the GoTaq ® qPCR Master Mix kit (Promega, Madison, WI, USA), as well as 0.2 µM of forward 5′-GAT GAT CTA CTG AGA CTG TGT GA and reverse 5′-AGA GCT TCT ACA GCT GGG ACA primers [ 26 ]. Reaction conditions and negative controls were based on Basso et al [ 14 ] and Cruz et al [ 27 ]. Standard curve was used to express the number of DNA copies by adopting standardized plasmid diluted from 10 5 to 10 8 copies of DNA/µL, in duplicate.…”

“…Therefore, next-generation technologies should present a reliable, single-step target-virus detection mechanism and require minimum detection time. The limitation of these traditional tests lies in the need of having qualified professionals and specific equipment, which are often expensive and less applicable in resource-limited environments, such as small farms [ 13 , 14 ].…”

A nanobody‐horseradish peroxidase fusion protein‐based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2

“…Immunoperoxidase monolayer(IPMA), immunofluorescence assays (IFAs), and enzyme-linked immunosorbent assay (ELISA) are the most common diagnostic methods for detecting PCV-2 antibodies and the serum neutralization assay is the standard method for the detection of neutralizing antibodies. [ 22 , 33 , 34 ]. But IPMA or IFA assays required an aseptic technique and could take up to 72 h [ 35 ].…”

Plasmonic biosensors for food control

“…SPR biosensors can detect a specific biomarker of a pathogen even in a fmol range (Bhunia, 2008;Rasooly & Herold, 2006;Vidic et al, 2013). Compared to conventional microbiological methods, plasmonic methodology was proved easiest to perform and more affordable ($3 per sample was estimated for porcine circovirus 2 detection (Basso, Cruz, Silva, Pedrosa, & Araújo Junior, 2020),). Nevertheless, due to possible cross-reactivity of antibodies, which could lead to false positive results, in some cases, DNA probes are preferred as recognition elements.…”