Direct detection of unamplified spring viraemia of carp virus RNA using unmodified gold nanoparticles

Abstract

Spring viraemia of carp (SVC) is a viral disease that mainly affects carp Cyprinus carpio and other cyprinid fish, causing severe economic losses. Rapid detection and identification of spring viraemia of carp virus (SVCV) is crucial for effective disease management. Recent advances in nanoscience are having a significant impact on many scientific fields, especially biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection. Single-and double-stranded oligonucleotides can be adsorbed on gold nanoparticles (AuNPs) in colloidal solution under certain conditions. We exploited this phenomenon to develop a specific hybridization assay for direct detection of SVCV-RNA without prior amplification. The result of the hybridization process could be detected visually within 1 min when the colour of the reaction mixture changed from red to blue (positive reaction) or remains red (negative). The lower detection limit of the assay was estimated to be 10 −3 TCID 50 ml −1 SVCV-RNA, and it has the feasibility to detect the target virus-RNA in clinical specimens without previous amplification. In order to obtain an indication of the assay's performance on clinical samples we compared the optimized assay with nested RT-PCR in detection of SVCV-RNA in infected fish samples. The concordance of the 2 methods was defined as 100% when compared to nested RT-PCR positive and negative samples. The SVC-AuNPs assay requires only 15 min, eliminates the need for thermal cycling or detection instruments and is a specific and rapid tool for detection of SVCV-RNA directly from clinical samples. KEY WORDS: SVCV · AuNPs · Colorimetric detection · Diagnosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 100: [3][4][5][6][7][8][9][10] 2012 rescence, immunoperoxidase, and ELISA (Amos 1985, OIE 2009). Rapid ad vances in molecular biology techniques have led to the development of several molecular methods for SVCV detection, including hybridization assays, RT-PCR, nested RT-PCR (nRT-PCR) and real-time PCR (Oreshkova et al. 1999, Koutná et al. 2003, Yue et al. 2008. Although these assays are specific and sen sitive, they are time consuming and require sophis ticated apparatus and complex post-run manipu lations.Recently, noble metal nanoparticles, particularly gold nanoparticles (AuNPs), have been introduced as a promising approach to the development of the next generation of diagnostic assays (Mirkin et al. 1996, Storhoff et al. 2000. AuNPs have become an important alternative as imaging agents due to their noncytotoxicity, facile immunotargeting and nonsusceptibility to photobleaching or chemical/thermal de naturation, a problem commonly associated with dyes (Jain et al. 2006). Advances in functionalizing particles with oligonucleotides and tailoring their surface properties have paved the way to design a series of new and practical systems for nucleic acid detection (Mirkin et al. 1996, Elghanian et al. 1997, Storhoff et al. 2000, Daniel & Astru...

Spring viraemia of carp virus: recent advances

“…The specificity of the assay was 100% when compared with positive and negative nested RT-PCR samples (Saleh et al, 2012). This assay offers several advantages over PCR-based assays, as it does not require a thermal cycling instrument and has a shorter test time (only 15 min) (Saleh et al, 2012).…”

“…Saleh et al (2012) exploited this phenomenon to develop a specific hybridization assay for direct detection of SVCV from clinical specimens without prior amplification of viral RNA (Saleh et al, 2012). The specificity of the assay was 100% when compared with positive and negative nested RT-PCR samples (Saleh et al, 2012).…”

Gold Nanoparticles as a Potential Tool for Diagnosis of Fish Diseases

“…Oligonucleotide probes targeting specifi c sequences of a particular gene of interest of the pathogen. For example, we have recently described the development of a 26 bp specifi c probe (5′-GTC TAT CAT CAG CTA CAT CGC ATT CC-3′) designed to target the spring viremia of carp virus (SVCV) glycoprotein gene [ 29 ]. Assess the specifi city of the probe against other common aquatic pathogens sequences deposited in GenBank.…”

“…Extracted nucleic acids of the pathogens (DNA or RNA): in this illustrative protocol we use RNA extracted from SVCV grown in epithelioma papulosum cyprinid culture (EPC) [ 29 ] ( see Note 1 ).…”

“…We give an example based on the detection of unamplifi ed SVCV RNA that we recently described [ 29 ]. The assay is performed as follows:…”