Nervous necrosis virus (nodavirus) has been responsible for mass mortalities in aquaculture industry worldwide, with great economic and environmental impact. A rapid low-cost test to identify nodavirus genotype could have important benefits for vaccine and diagnostic applications in small- and medium-scale laboratories in both academia and fish farming industry. A dual lateral flow biosensor for simultaneous detection of the most prevalent nodavirus genotypes (RGNNV and SJNNV) was developed and optimized. The dual biosensor consisted of two antibody-based test zones, indicative of each genotype, and a control zone. The positive signals were visualized by gold nanoparticles functionalized with anti-biotin antibody, and the detection was completed within 20 min. Optimization studies included antibody type and amount determination for test zone construction, gold nanoparticle conjugate type selection for high signal generation, and detection assay parameter determination. Following optimization, the biosensor was evaluated with healthy and RGNNV-nodavirus-infected fish samples. The proposed assay’s cost was estimated to be less than 3 €, including the required reagents and biosensor. This work presents important steps towards making a dual lateral flow biosensor for nodavirus genotyping; further evaluation with clinical samples is needed before the test is appropriate for diagnostic kit development.
Diseases of viral etiology have been wreaking havoc in the aquaculture industry, which is considered of strategic importance for Greek, European, and worldwide economies. Viral diseases often wipe out entire stocks within days of onset of infection with major economic and environmental costs [
Nervous necrosis virus (NNV), also known as nodavirus, has been recognized as the causative agent of VNN. Fish nodavirus belongs to the Nodaviridae family and the
Nodaviruses belonging to different genotypes have different host ranges [
Analysis of nodavirus genetic variation would vastly benefit the rational development of effective vaccines and diagnostic reagents. Molecular methods such as polymerase chain reaction (PCR) are extensively used for nodavirus detection [
Lateral flow paper biosensors (LFB) provide a tool, which is ideal for sensitive, reproducible, and accurate detection of PCR products, in a rapid way, implanted successfully in research laboratory setups. LFBs are prefabricated paper strips containing dry reagents that are activated by applying a sample-containing solution. They are designed for disposable single use and for applications where an on/off signal is sufficient [
To further facilitate nodavirus genotyping with a promising technique, the aim of the present study was the development and optimization of a dry-reagent lateral flow biosensor for simultaneous visual detection of two different nodavirus genotypes, namely RGNNV and SJNNV. The dry-reagent biosensor was prepared by selecting the proper antibodies and optimizing their deposited amounts. Next, gold nanoparticles, which serve as signal reporters, were modified by conjugation with anti-biotin antibody. In a proof-of-principle test, viral samples were prepared by extracting RNA from healthy and infected fish samples and subjected to tetra-primer PCR for simultaneous amplification of SJNNV and RGNNV genotypes. Application of PCR products on functional dual lateral flow biosensor allowed detection of the genotype of the present virus by naked eye (visual). Knowledge of the correct nodavirus genotype is a valuable tool allowing more effective diagnosis and treatment of disease pathologies.
Synthesis of the oligonucleotides used in the present study was performed by Eurofins Genomics AT (Vienna, Austria). The primers were designed with respect to the publicly available RGNNV and SJNNV genotype sequences (GenBank accession numbers: Y08700.1 and NC_003449.1, resp.), as described before [
Two reference plasmids, specific for each genotype (GenScript, Piscataway, NJ, USA), were used as targets for tetra-primer PCR optimization studies. The sequences of the pRGNNV and pSJNNV are described in detail in [
The principle of the dual lateral flow biosensor is illustrated schematically in Figure
Principle of the dual nanoparticle-based lateral flow biosensor for simultaneous detection of nodavirus SJNNV and RGNNV genotypes. Two test zones (anti-fluorescein antibody (TZ-R) and anti-digoxigenin antibody (TZ-S)) and a control zone (biotinylated BSA (CZ)) have been deposited on the diagnostic membrane. The sample, containing the respective genotype of the target analyte (tetra-primer PCR product with fluorescein (F) for RGNNV genotype or digoxigenin (D) for SJNNV genotype), is hybridized with a biotinylated genotype-specific oligonucleotide probe and applied on the conjugation pad, where functionalized gold nanoparticles (Au) with anti-biotin antibody have already been added. Following that, the biosensor is immersed in the developing buffer, the sample and the nanoparticles are immobilized on the appropriate test zone, and the positive signal is visible by the naked eye, as a red zone. The excess nanoparticles bind to the control zone of the biosensor. The image shows a side view of the lateral flow biosensor. IP: immersion pad; CP: conjugation pad; M: diagnostic membrane; AP: absorbent pad. The assay components are not in scale.
Gold nanoparticle (Au NP) functionalization with anti-biotin antibody was performed following the previously described protocol [
The preparation of anti-BSA gold nanoparticles, as described in [
The antibody-gold nanoparticle conjugates were stored at 4°C.
The dual dry reagent lateral flow biosensors (4 × 60 mm) were prepared as described before in [
All samples used in the present study were European sea bass (
The RNeasy Mini kit (Qiagen, Hilden, Germany) was used for total RNA extraction, according to the manufacturer’s instructions. Measurements of the absorbance at 260 nm (
The purified total RNA was reverse transcribed (RT) with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). The RT reaction consisted of 0.5 mM dNTPs (dNTPs: dATP, dTTP, dCTP, dGTP; HT Biotechnology, Cambridge, UK), 2.5
The tetra-primer PCR amplification was performed with GoTaq Flexi DNA polymerase (0.625 units; Promega, WI, USA) in GeneAmp PCR System 9700 cycler (Applied Biosystems, NY, USA). The reaction mixtures contained 1 × GoTaq Flexi Buffer, 200
Four reference oligonucleotides were utilized as target sequences: oligonucleotides B-dA20 and B-dC20 were designed to contain a biotin molecule in their 5′ end, in order to interact with Au NPs functionalized with anti-biotin antibody. The oligonucleotide dig-dT20 was designed with a digoxigenin molecule in its 5′ end in order to be immobilized by the anti-digoxigenin antibody (TZ-S zone) while the oligonucleotide fluor-dG20 was designed with a fluorescein molecule in its 5′ end to interact with immobilized anti-fluorescein antibody (TZ-R zone). Two target mixtures were prepared: Dig-mixture consisted of 1 pmol B-dA20, 1 pmol dig-dT20, and ddH2O; Fluor-mixture consisted of 1 pmol B-dC20, 1 pmol fluor-dG20, and ddH2O. The mixtures were denatured at 95°C, for 3 min, and left to hybridize at 37°C for 10 minutes. Five microlitres of each target mix was applied on the LFBs. Next to them, 10
For the TZ-R zone with monoclonal anti-fluorescein antibody, a solution consisting of 500 mg/L anti-fluorescein antibody (Millipore, Billerica, MA, USA), 50 mL/L methanol, and 20 g/L sucrose in freshly prepared 100 mM NaHCO3 buffer (pH 8.5) was loaded at a density of 500 ng per LFB. For the TZ-R zone with polyclonal anti-fluorescein antibody, 500 mg/L anti-fluorescein antibody (Meridian, Memphis, TN, USA) were mixed with the abovementioned buffer and loaded at a density of 500 ng per LFB. The procedures were performed as described in Section
In order to perform the antibody amount optimization studies, two mixes were prepared for each amount; that is, for TZ-S zone construction, two solutions were prepared: Solution 1 contained 250 mg/L while solution 2 contained 500 mg/L of anti-digoxigenin antibody diluted in 50 mL/L methanol and 20 g/L sucrose in 100 mM NaHCO3 buffer (pH 8.5). The TZ-R zone construction was tested with solution 3, consisting of 75 mg/L of polyclonal anti-fluorescein antibody or 500 mg/L of the same antibody (solution 4) in 50 mL/L methanol, and 20 g/L sucrose in freshly prepared 100 mM NaHCO3 buffer (pH 8.5).
The target mixtures were prepared as before (Section
Detection of the nodavirus tetra-primer PCR products was performed as follows: aliquots of PCR solutions were mixed with 90 mM NaCl, 1 pmol of each biotin-labelled genotype-specific nodavirus probe and 1 × PCR buffer (final volume: 5
The dual LFB assay for tetra-primer PCR RGNNV- and SJNNV-specific product detection was optimized by comparing the detection specificity and efficiency obtained, using (i) different amounts of oligonucleotide detection probes (0.5–4 pmol/1 pmol of target; i.e., 0.5/1/2, and 4 pmol of probes) and (ii) different annealing temperatures (i.e., 25, 37, and 42°C). The parameter that resulted in the highest amount of specific signal in the appropriate test zone and the smallest amount of nonspecific signal was chosen as the optimum condition in each case.
The proposed dual biosensor format was developed by our research group and has been successfully exploited on pharmacogenetic studies for cytochrome c single nucleotide polymorphism genotyping, combined with oligonucleotide ligation reaction [
The proposed dual LFB consisted of two test lines made by anti-digoxigenin (TZ-S) and anti-fluorescein antibodies (TZ-R) and a control zone which was made by biotinylated BSA, absorbed by the membrane. The signal visualization was realized by Au NPs conjugated with anti-biotin antibodies. The anti-digoxigenin antibody performance for test zone construction was evaluated in the previously mentioned study [
Dual lateral flow biosensor optimization studies. Representative lateral flow biosensors and signal intensity graphs for (a) effect of the use of monoclonal (mAnti-fluor) versus polyclonal (pAnti-fluor) anti-fluorescein antibody; (b) effect of the anti-digoxigenin (anti-dig) antibody amount for test zone construction; and (c) effect of the polyclonal anti-fluorescein antibody amount for test zone construction. All tests were performed with dig- and fluor-reference target mixtures. Signal is visualized with anti-biotin Au NPs. TZ-S: anti-digoxigenin zone; TZ-R: anti-fluorescein zone.
The immobilized anti-fluorescein and anti-digoxigenin antibody amounts were examined next. Two concentrations of each antibody were tested. The amount of anti-digoxigenin antibody on the TZ-S was initially studied (Figure
Recently, our research group developed a signal amplification methodology in one-step for nucleic acid detection lateral flow biosensors based on gold nanoparticles [
Effect of anti-biotin functionalized gold nanoparticle amount and signal enhancement with nanoparticle aggregates. Representative lateral flow biosensors and signal intensity graphs for Dig- and Fluor-reference target mixtures. Signal is visualized with (1) 30 nm gold nanoparticles functionalized with anti-biotin antibodies (5
Optimization studies for assessment of the oligonucleotide probe impact in the hybridization reaction mixtures were performed. The oligonucleotide probes were tested in amounts of 0.5–4 pmol/1 pmol of target (Figures
Dual lateral flow biosensor assay optimization studies. Representative lateral flow biosensors and signal intensity graphs for (a) oligonucleotide probe amount effect on plasmid pRGNNV amplification product hybridization mixture; (b) oligonucleotide probe amount effect on plasmid pSJNNV amplification product hybridization mixture; (c) hybridization temperature effect on the pRGNNV PCR product and specific oligonucleotide probe hybridization; and (d) hybridization temperature effect on the pSJNNV PCR product and specific oligonucleotide probe hybridization. The signal is visualized with anti-biotin Au NPs. TZ-S: anti-digoxigenin zone; TZ-R: anti-fluorescein zone.
The hybridization temperature effect on target PCR product and specific oligonucleotide probe hybridization was tested with a temperature of 25–42°C. As observed in Figures
The dual lateral flow assay reproducibility was assessed since it is one of the most important parameters for successful biosensor development. The proposed assay reproducibility was assessed with simultaneous application of the Dig- and Fluor-reference mixtures on the dual biosensors. Six biosensors which were prepared in different batches (i.e., LFBs 1 and 2: batch 1; LFBs 3 and 4: batch 2; and LFBs 5 and 6: batch 3) were tested with the reference mixtures, and the test and control zones intensities were measured. The results are presented in Figure
Reproducibility study. (a) Typical images of lateral flow biosensors with anti-biotin-gold nanoparticle conjugates after applying Dig- and Fluor-reference target mixtures as targets. (b) Optical density of the test and control zones. Graph of the intensities of the lateral flow biosensor test and control zones for reproducibility assessment (CVTZ-R: 3.9%; CVTZ-s: 8.2%; CVCZ: 9.2%,
The dual lateral flow biosensor was used to detect amplification products of both genotype-specific plasmids (pRGNNV and pSJNNV), one nodavirus infected
Visual detection of nodavirus genotype-specific products with dual lateral flow biosensors. Representative LFBs with amplification products of tetra-primer PCR performed with pRGNNV and pSJNNV reference plasmids and a healthy (S_1) and an infected (S_2)
As mentioned in our previous studies [
The proposed dual lateral flow biosensor constitutes a step forward to a robust, rapid, and accurate tool for fish virus genotype assessment with ease and low cost. The assay can be utilized as a potential detection system for virus genotyping by small- and medium-size research labs and the aquaculture industry, providing the means for effective vaccine and diagnostic development. The results demonstrate the optimization studies for a rapid single-step assay, which requires low amount of the analyzed sample and provides simultaneous amplification and genotyping of nodavirus DNA in a single, closed-tube methodology. The assay was optimized in terms of the biosensors’ preparation and the detection assay parameters, demonstrating attractive characteristics with respect to specificity and reproducibility. The optimum goal for the proposed methodology is to replace the costly sequencing for virus genotyping, since such simple-to-use and low-cost methods are ideal for medium-scale laboratories.
The main advantage of the proposed method compared with previously used methods (i.e., gel electrophoresis and melting analysis) is that the dual biosensor minimizes the need for specialized and costly instrumentation and reagents. Therefore, it enables rapid and low-cost genotyping of nodavirus by visual detection of the RGNNV/SJNNV amplification product. Also, the tetra-primer PCR product is directly hybridized with genotype-specific probes without prior purification from the excess of primers and dNTPs, and the hybridization mixture is applied on the biosensors’ conjugate pad, minimizing the possibility for contamination. Use of the genotype-specific probe and product detection by hybridization provides extra sequence confirmation, in contrast with electrophoresis that provides only the size of the amplification products. The visual detection of the genotype-specific product is completed in 20 min, and the overall assay can provide a samples’ genotype in less than 4 hours. Finally, the lateral flow biosensor format minimizes the requirements for highly qualified personnel for performing the test and interpreting the results.
The authors declare that there are no conflicts of interest regarding the publication of this article. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
Dimitra K. Toubanaki conceived, designed, and performed the experiments; Dimitra K. Toubanaki and Evdokia Karagouni analyzed the data; Dimitra K. Toubanaki wrote the paper; Evdokia Karagouni proofread the manuscript. All authors have approved the present manuscript.
The research project was implemented within the framework of the Action “Supporting Postdoctoral Researchers” of the Operational Program “Education and Lifelong Learning” (Action’s Beneficiary: General Secretariat for Research and Technology) and was cofinanced by the European Social Fund (ESF) and the Greek State.