Development of functionalized gold nanoparticles as nanoflare probes for rapid detection of classical swine fever virus
Graphical abstract
Introduction
Classical swine fever (CSF) is a highly contagious disease affecting swine that is caused by classical swine fever virus (CSFV); it is classified as an OIE List A disease that has caused major damages to the swine industry [1]. Although some countries of the European Union have had some success in controlling the epidemic of CSF, infection by CSFV causes a major disease that endangers the pig-breeding industries of developing countries, including China [2]. Eradicating CSF is difficult because methods are still needed for both rapid and effective diagnosis and the monitoring of the occurrence and spread of CSF. CSF is characterized by hemorrhagic lymphadenitis, high fever, depression, and diffuse hemorrhaging of the skin, kidneys, and other organs. Although those symptoms are easily identified in a clinical diagnosis, CSFV is usually considered to be a potential infection in pigs before relying on clinical symptoms to rule out CSF [3]. Thus, the rapid detection of CSFV is of great significance for the diagnosis, early warning, and prevention of CSF.
Considering the hazards to the global pig-breeding industry caused by CSFV infection, researchers have performed exploratory research on detection technologies for CSFV, mainly based on three detection mechanisms. (1) Observation of virions. The presence of virus particles can be directly observed by electron microscopy, a technique that is regarded as the “gold standard” for virus detection [4]. The advantage of this technology is its direct detection of CSFV, but the equipment is expensive and observation is limited to research institutions. (2) Immunodiagnostic technique. As an antigen, CSFV can stimulate the host’s immune system to produce antibodies and also be neutralized by specific antibodies. Based on the characteristics of antigen–antibody binding, a number of methods for the detection of CSFV have been designed that utilize the enzyme-linked immunosorbent assay (ELISA) approach [5,6]. However, the high cost of antibody preparation and false-negative results are frequent problems preventing the use of this method to inspect a herd. (3) Detection of viral nucleic acid. As a pestivirus within the family Flaviviridae, CSFV has a single-stranded RNA genome of approximately 12,300 bases that can be detected soon after infection. Therefore, researchers have detected specific CSFV RNA sequences using different techniques in recent years, such as the polymerase chain reaction (PCR) [7] and quantitative polymerase chain reaction (qPCR) [8]. These methods quantitatively analyze viral nucleic acid by detecting specific nucleotides and greatly improve the sensitivity of these methods for the detection of CSFV. The problem with these approaches is the complication of extracting RNA from the diseased specimen; polluted or degraded RNA often yields false positive or false negative results.
Considering the continued risk of CSF to swine populations, an assay to identify and monitor this disease is urgently needed. Researchers have also realized that different methods have different limitations. Future detection methods for CSFV should be able to directly detect the virion with high sensitivity and wide availability. Gold nanoparticles (AuNPs) are widely used in biological imaging and other fields as target carriers [9], enhancers [10,11], and tracers [12,13] with special surface plasmon resonance properties, good biocompatibility, and high fluorescence quenching. Therefore, this work represents the latest international research progress in nanotechnology to establish a nanoflare model that achieves a breakthrough in the detection of CSFV. Specific RNA sequences of CSFV were demonstrated by fluorescence enhancement-based detection by the nanoflare. The detection of CSFV was successfully shown in CSFV-infected macrophages as well as different pathological tissue samples from a CSFV-positive pig, without using virus nucleic acid amplification. Our work facilitates the application of nanomaterials in virus detection for veterinary medicine.
Section snippets
Synthesis of nanoflares
Two oligonucleotides, a recognition strand (5′-GUUGATGATATTGCGTACCUGAAAAAA-Thiol Modifier C3-S-3′) and a reporter strand (5′-Cy3-TCAGGTACGCAA-3′), were synthesized by Genscript Biotech Corporation (Suzhou, China). The two oligonucleotides were hybridized at a 1:1.2 M ratio in phosphate-buffered saline (PBS). These oligonucleotides were heated at 75℃ for 0.5 h, and then the sample was cooled at room temperature while avoiding light to permit hybridization [14]. AuNPs were synthesized through the
Results and discussion
In this study, oligonucleotide-AuNP conjugates that possessed a highly specific sequence, designed and synthesized according to the target virus sequence, were developed to achieve real-time, quantitative detection of CSFV antigens. In our nanocomposite sensor for the detection of CSFV, AuNPs were conjugated with a pair of complementary DNA sequences, the recognition strand and reporter strand (Fig. 1A). The recognition strand was a thiolated CSFV-recognizing sequence, while the reporter strand
Conclusions
The present study illustrates the application of oligonucleotide-AuNP nanotechnology in obtaining a CSFV-AuNP probe that provides rapid, specific, sensitive, and accurate determination of the CSFV nucleic acid in lab and field samples, without virus nucleic acid amplification. This work facilitates early warning, diagnosis, and prevention of CSF and provides a nanoflare technology as a reference for virus detection in animal husbandry.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (31470535), the National 1000 Young Talents Program of China and National Natural Science Foundation of Shaanxi Province under Grant (No. 2018JM2041). Thanks to Mr. Chengbo Wang for collecting samples.
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