High-throughput Sample Analysis Research Articles

Lensfree dynamic super-resolved phase imaging based on active micro-scanning.

In this Letter, we present a new active micro-scanning-based imaging platform and associated super-resolution (SR) phase retrieval method in lensfree microscopy to achieve SR dynamic phase imaging. The samples are illuminated by a nearly coherent illumination system, where two orthogonal parallel plates are inserted into the light path and rotate to achieve controllable source micro-scanning, permitting sub-pixel shifts of the holograms on x- and y-axis directions independently. Then sequential low-resolution sub-pixel-shifted holograms are processed to enhance spatial resolution and reconstruct quantitative phase images of the sample simultaneously. The reconstruction result of the benchmark quantitative phase microscopy target (QPTTM) demonstrates a half-pitch lateral resolution of 775nm across a large field-of-view of ∼29.84 mm2, surpassing 2.15 times that of the theoretical Nyquist-Shannon sampling resolution limit imposed by the pixel size of the imaging sensor (1.67μm). The proposed approach is also evaluated by imaging unstained HeLa cells, suggesting it is a promising toolset for high-throughput monitoring and quantitative analysis of unlabeled biological samples.

Antibiotic analysis using Electro‐Filtering Paper Spray Ionization

In this work, we analyzed the performance of the Electro-Filtering Paper Spray Ionization (EFSI) method for detecting compounds in unprocessed samples. A relatively rigid and electrically conductive copper filter was used as a substrate to insure sufficient and efficient sample-solvent extraction and to increase the conductivity for paper spraying. The method was demonstrated as applicable for indirect high-throughput analysis of large-volume unprocessed samples, which is not possible with conventional nanoESI or direct paper spray methods. The new method can generate different desired ion signals for a wide range of compounds by selecting different extraction solvents. Moreover, key parameters related to extraction efficiency were optimized in detail to obtain the most satisfactory extraction efficiency during antibiotic analysis. Finally, under optimal conditions, the EFSI method was successfully used to detect four antibiotics in animal products of egg, chicken, and chicken liver, exhibiting good reproducibility with calibration curves between 81.6% and 96.3%, and R2 values above 0.99. Recoveries of 75.0%ￚ94.6% were obtained for the four antibiotics. Hence, the proposed EFSI-MS is a successful, economical, rapid, and high-throughput method that is effective for both unknown and targeted extraction of unprocessed samples by mass spectrometric analysis.

Development and validation of a high-throughput assay for the quantification of multiple green tea-derived catechins in human plasma.

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra-batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high-throughput sample analysis studies.

SDS-PAGE fractionation to increase metaproteomic insight into the taxonomic and functional composition of microbial communities for biogas plant samples.

Metaproteomics represent an important tool for the taxonomic and functional investigation of microbial communities in humans, environment, and technical applications. Due to the high complexity of the microbial communities, protein, and peptide fractionation is applied to improve the characterization of taxonomic and functional composition of microbial communities. In order to target scientific questions regarding taxonomic and functional composition adequately, a tradeoff between the number of fractions analyzed and the required depth of information has to be found. Two samples of a biogas plant were analyzed by either single LC-MS/MS measurement (1D) or LC-MS/MS measurements of fractions obtained after SDS-PAGE (2D) separation. Fractionation with SDS-PAGE increased the number of identified spectra by 273%, the number of peptides by 95%, and the number of metaproteins by 59%. Rarefaction plots of species and metaproteins against identified spectra showed that 2D separation was sufficient to identify most microbial families but not all metaproteins. More reliable quantitative comparison could be achieved with 2D. 1D separation enabled high-throughput analysis of samples, however, depth in functional descriptions and reliability of quantification were lost. Nevertheless, the proteotyping of multiple samples was still possible. 2D separations provided more reliable quantitative data combined with a deeper insight into the taxonomic and functional composition of the microbial communities. Regarding taxonomic and functional composition, metaproteomics based on 2D is just the tip of an iceberg.

Development of an automated high-throughput sample preparation protocol for LC-MS/MS analysis of glycated peptides

Advanced glycation end products (AGEs) are known to play a leading part in the pathogenesis of human diseases, such as diabetes, Alzheimer's disease, lateral sclerosis, and atherosclerosis. It is for this reason that research on AGEs is crucial and large-scale studies are needed for the treatment of diseases. The aim of this study was to develop a reproducible method to analyze Amadori compounds using an automated enrichment protocol for high-throughput analysis of clinical samples. The developed method enabled the enrichment of Amadori compounds simultaneously from 96 samples, and it was applied to the discovery of biomarkers in AGEs related diseases. In this study, ten human serum samples were processed using automated filter-aided sample preparation (aFASP) in a 96-well filter plate, and the eluted peptide mixtures were enriched by Affinity Cellufine PB in a fritted 96-well filter plate using a liquid handling robotic system. The eluted glycated peptides were analyzed by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. A total of 982 unique glycated peptides, corresponding to 524 unique glycated proteins, were identified from the analysis of ten human samples. The advantages and potentials of the automated sample preparation system were demonstrated through label free quantification of the glycated peptides.

RETRACTED: Proteomic studies on lactic acid bacteria: A review

Probiotics are amongst the most common microbes in the gastro-intestinal tract of humans and other animals. Prominent among probiotics are Lactobacillus and Bifidobacterium. They offer wide-ranging health promoting benefits to the host which include reduction in pathological alterations, stimulation of mucosal immunity and interaction with mediators of inflammation among others. Proteomics plays a vital role in understanding biological functions of a cell. Proteomics is also slowly and steadily adding to the existing knowledge on role of probiotics. In this paper, the proteomics of probiotics, with special reference to lactic acid bacteria is reviewed with a view to understand i) proteome map, ii) mechanism of adaptation to harsh gut environment such as low pH and bile acid, iii) role of cell surface proteins in adhering to intestinal epithelial cells, and iv) as a tool to answer basic cell functions. We have also reviewed various analytical methods used to carry out proteome analysis, in which 2D-MS and LC-MS/MS approaches were found to be versatile methods to perform high-throughput sample analyses even for a complex gut samples. Further, we present future road map of understanding gut microbes combining meta-proteomics, meta-genomics, meta-transcriptomics and -metabolomics.

A Versatile Integrated Ambient Ionization Source Platform.

The pursuit of high-throughput sample analysis from complex matrix demands development of multiple ionization techniques with complementary specialties. A versatile integrated ambient ionization source (iAmIS) platform is proposed in this work, based on the idea of integrating multiple functions, enhancing the efficiency of current ionization techniques, extending the applications, and decreasing the cost of the instrument. The design of the iAmIS platform combines flowing atmospheric pressure afterglow (FAPA) source/direct analysis in real time (DART), dielectric barrier discharge ionization (DBDI)/low-temperature plasma (LTP), desorption electrospray ionization (DESI), and laser desorption (LD) technique. All individual and combined ionization modes can be easily attained by modulating parameters. In particular, the FAPA/DART&DESI mode can realize the detection of polar and nonpolar compounds at the same time with two different ionization mechanisms: proton transfer and charge transfer. The introduction of LD contributes to the mass spectrometry imaging and the surface-assisted laser desorption (SALDI) under ambient condition. Compared with other individual or multi-mode ion source, the iAmIS platform provides the flexibility of choosing different ionization modes, broadens the scope of the analyte detection, and facilitates the analysis of complex samples. Graphical abstract ᅟ.

Omics research project on prospective cohort studies from the Tohoku Medical Megabank Project.

Population-based prospective cohort studies are indispensable for modern medical research as they provide important knowledge on the influences of many kinds of genetic and environmental factors on the cause of disease. Although traditional cohort studies are mainly conducted using questionnaires and physical examinations, modern cohort studies incorporate omics and genomic approaches to obtain comprehensive physical information, including genetic information. Here, we report the design and midterm results of multi-omics analysis on population-based prospective cohort studies from the Tohoku Medical Megabank (TMM) Project. We have incorporated genomic and metabolomic studies in the TMM cohort study as both metabolome and genome analyses are suitable for high-throughput analysis of large-scale cohort samples. Moreover, an association study between the metabolome and genome show that metabolites are an important intermediate phenotype connecting genetic and lifestyle factors to physical and pathologic phenotypes. We apply our metabolome and genome analyses to large-scale cohort samples in the following studies.

Real-time PCR, a great tool for fast identification, sensitive detection and quantification of important plant-parasitic nematodes

Plant-parasitic nematodes can cause significant damage to agricultural crops and forests worldwide, resulting in major economic losses. Some nematode species do not occur in all areas and are regulated as quarantine organisms. To avoid introduction and spread of these organisms, fast, simple and reliable detection and identification methods are needed, that help plant diagnostic services such as reference centres or national plant protection organizations (NPPOs) to rapidly identify suspicious nematodes. Real-time PCR is one of the fastest, most sensitive and reliable methods to fulfil this task. It is a DNA-based method that is easy to learn with the only requirement of having a specific thermocycler (Real-time Platform) and the appropriate chemistry. Real-time PCR provides very sensitive detection and species-specific identification with the potential to quantify target organisms if required. Following DNA extraction, results can be seen in 1–3 h and management decisions applied. Real-time PCR can be used for high-throughput analysis of many samples and in some cases for multiplexing, allowing for identification of more than one species in a single reaction. Over the past 15 years, real-time PCR methods have been developed for the main plant-parasitic nematodes, in particular the regulated species. This paper reviews the achievements in plant nematology diagnostics using real-time PCR as the method of choice for fast and reliable detection, identification and even quantification of plant parasitic nematodes.

Analytical characteristics of nano-electrospray operated under super-atmospheric pressure

High-pressure nanoelectrospray ionization (nanoESI) source is a recently developed technique in which the electrospray ionization is generated inside an enclosed chamber with gas pressure higher than the atmospheric pressure. In this paper, the performance of nanoESI under different gas pressures, emitter position, ion inlet temperature, additive for desalination are presented. Under a pressure of 2 bars, the nanoESI is almost eased from the electrical discharge problem, and that offers a wider tuning window for the emitter potential to produces a higher and more stable ion signal. With optimized ion inlet temperature, the high-pressure operation facilitates the generation of ion species of higher charge-state from the highly aqueous solution, and produced less sodium adducts. A preparation method for the high-throughput analysis of raw biological samples using disposable plastic nanoESI emitter is also described.

UHPSFC/ESI-MS Analysis of Lipids.

This new analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is based on lipid class separation using 1.7μm particle bridged ethylene hybrid silica columns and a gradient of methanol-water-ammonium acetate mixture as a modifier. The method enables a fast separation of 30 nonpolar and polar lipid classes within 6-min analysis time covering six main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive- and negative-ion full scan and tandem mass spectra measured with high mass accuracy and high resolving power. The method is used for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive, and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in clinical research.

Studies on the development of antibodies for the highly hydrophobic plasticizers DINCH and DEHT

Diisononylcyclohexane-1,2-dicarboxylate (DINCH) and di-2-ethylhexyl terephthalate (DEHT), two of the most important substitutes for phthalate plasticizers, are used for a wide range of applications. Consequently, an increasing occurrence in urine and environmental samples is reported. Reliable and fast analytical methods for the quantification of these plasticizers are needed. So far, mainly GC-MS or LC-MS methods are used. We aimed to develop the first antibodies and immunoassays allowing for high-throughput analysis of samples. We designed two DINCH hapten structures and one DEHT hapten structure and employed hapten-protein conjugates for the immunization of rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISAs) against each hapten using the produced polyclonal antibodies were established. Yet, binding of DINCH to the respective antibodies was not observed in neither direct nor indirect assay formats, even when using protein conjugates with the heterologous haptens and different carrier proteins in the indirect format. The use of surfactants and solvents in the sample buffer did not result in recognition of the plasticizers. Also, no binding of DEHT in ELISA employing the respective antibodies was detected. We speculate that the production of antibodies against these highly hydrophobic molecules is not possible via our route, however a different hapten design could overcome this obstacle.

Automated Comparative Metabolite Profiling of Large LC-ESIMS Data Sets in an ACD/MS Workbook Suite Add-in, and Data Clustering on a New Open-Source Web Platform FreeClust.

The technological development of LC-MS instrumentation has led to significant improvements of performance and sensitivity, enabling high-throughput analysis of complex samples, such as plant extracts. Most software suites allow preprocessing of LC-MS chromatograms to obtain comprehensive information on single constituents. However, more advanced processing needs, such as the systematic and unbiased comparative metabolite profiling of large numbers of complex LC-MS chromatograms remains a challenge. Currently, users have to rely on different tools to perform such data analyses. We developed a two-step protocol comprising a comparative metabolite profiling tool integrated in ACD/MS Workbook Suite, and a web platform developed in R language designed for clustering and visualization of chromatographic data. Initially, all relevant chromatographic and spectroscopic data (retention time, molecular ions with the respective ion abundance, and sample names) are automatically extracted and assembled in an Excel spreadsheet. The file is then loaded into an online web application that includes various statistical algorithms and provides the user with tools to compare and visualize the results in intuitive 2D heatmaps. We applied this workflow to LC-ESIMS profiles obtained from 69 honey samples. Within few hours of calculation with a standard PC, honey samples were preprocessed and organized in clusters based on their metabolite profile similarities, thereby highlighting the common metabolite patterns and distributions among samples. Implementation in the ACD/Laboratories software package enables ulterior integration of other analytical data, and in silico prediction tools for modern drug discovery.

Engineered Paper-Based Cell Culture Platforms.

Paper is used in various applications in biomedical research including diagnostics, separations, and cell cultures. Paper can be conveniently engineered due to its tunable and flexible nature, and is amenable to high-throughput sample preparation and analysis. Paper-based platforms are used to culture primary cells, tumor cells, patient biopsies, stem cells, fibroblasts, osteoblasts, immune cells, bacteria, fungi, and plant cells. These platforms are compatible with standard analytical assays that are typically used to monitor cell behavior. Due to its thickness and porous nature, there are no mass transport limitations to/from the cells in paper scaffolds. It is possible to pattern paper in different scales (micrometer to centimeter), generate modular configurations in 3D, fabricate multicellular and compartmentalized tissue mimetics for clinical applications, and recover cells from the scaffolds for further analysis. 3D paper constructs can provide physiologically relevant tissue models for personalized medicine. Layer-by layer strategies to assemble tissue-like structures from low-cost and biocompatible paper-based materials offer unique opportunities that include understanding fundamental biology, developing disease models, and assembling different tissues for organ-on-paper applications. Paper-based platforms can also be used for origami-inspired tissue engineering. This work provides an overview of recent progress in engineered paper-based biomaterials and platforms to culture and analyze cells.

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 µm, C8 100 Å; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 µm, C8 100 Å; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Culex torrentium mosquitoes from Germany are negative for Wolbachia.

Wolbachia (Rickettsiales: Anaplasmataceae) infects a wide range of arthropods, including several mosquito species. The bacterium is known to induce a plethora of phenotypes in its host, examples being the reproductive phenotype cytoplasmic incompatibility or resistance against infection with arboviruses. The latter is especially relevant when assessing the vector competence of mosquito species for emerging arboviruses. Thus, knowledge of Wolbachia infection status is important for the assessment of vector competence. To facilitate Wolbachia screening in mosquito populations, a quantitative polymerase chain reaction (qPCR) assay was developed to enable high-throughput analysis of mosquito samples. Using this assay, the Wolbachia infection status of the two most common Culex mosquito species in Germany, Culex pipiens biotype pipiens Linnaeus (Diptera: Culicidae) and Culex torrentium Martini (Diptera: Culicidae), was assessed. About 93% of all tested C. pipiens biotype pipiens individuals were positive for Wolbachia, whereas none of the C. torrentium samples was found to be infected. Furthermore, other applications of the qPCR assay were explored by assessing a potential link between the levels of Wolbachia and West Nile virus (WNV) infections in German C. pipiens biotype pipiens mosquitoes. No relationship was found between the two variables, indicating that a Wolbachia-induced antiviral phenotype in this mosquito population is not exclusively attributable to the general level of bacterial infection.

New insights to improve resolution and reliability of Raman spectral analysis using higher-density multiscale regression

When paired with sophisticated multivariate calibration methods, spectroscopy techniques stand out conspicuously for high-throughput analysis of complex samples. However, despite the identification capability of multivariate analysis, fluctuant spectral interference and complicated chemometrics procedure often combine to limit conventional spectroscopy techniques. In this regard, we proposed a novel strategy, higher-density multiscale regression (HDMR), to adaptively process Raman spectra for quantitative analysis. HDMR firstly splits the Raman spectra into frequency components at different scales using higher-density discrete wavelet transform (HDDWT), yielding a larger number of subbands to benefit a good separation of spectral bands. Parallel member models constructed with these decomposed components are then fused into a final prediction through partial least square (PLS) reweighting strategy. With HDMR, the pre-treatment process and multivariate calibration are integrated into a unified calibration model for Raman spectra at hand, regardless of its structural characteristics. This would definitely avoid of information leakage in multivariate calibration, thus promoting the application of Raman spectroscopy technique as a general tool for analytical chemistry. To validate the performance of the HDMR, two industrial Raman spectral data sets were investigated to yield challenges representative of those encountered in Raman spectroscopy. This proposed strategy has improved calibration performance through the reweighting way. Satisfactory calibration results suggest that HDMR provides a universal tool for reliable Raman spectral analysis, which may well extend to other spectral data sets.

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

BackgroundCytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material.MethodsWe established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species.ResultsAs a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice.ConclusionsPlant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations.

Genotype-specific real-time PCR combined with high-resolution melting analysis for rapid identification of red-spotted grouper nervous necrosis virus

A real-time genotype-specific polymerase chain reaction (PCR) assay combined with high-resolution melting (HRM) analysis was developed to assess the most common genotypes of nervous necrosis viruses or nodaviruses. Nodaviruses are the causal agents of viral nervous necrosis infections, which have been wreaking havoc in the aquaculture industry worldwide, with fish mortality up to 100%. The four different genotypes of nodaviruses correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostics requires analysis of genetic variation among viruses. The aim of the present study was to develop a real-time tetra-primer genotype-specific PCR assay for genotype identification. Four primers were utilized for simultaneous amplification of nodavirus genotype-specific products in a single closed-tube PCR after a reverse-transcription reaction using RNA isolated from fish samples. For high-throughput sample analysis, SYBR Green-based real-time PCR was used in combination with HRM analysis. The assay was evaluated in terms of specificity and sensitivity. The analysis resulted in melting curves that were indicative of each genotype. The detection limit when using reference plasmids was 100 ag/µL for both genotypes, while the sensitivity of the assays when testing a complex mixture was 10fg/µL for red-spotted grouper nervous necrosis virus (RGNNV) and 100fg/µL for striped jack nervous necrosis virus (SJNNV). To test the capability of this method under real-world conditions, 58 samples were examined. All samples belonged to the RGNNV genotype, which was fully validated. The results were in full agreement with genotyping by reference methods. The proposed methodology provides a rapid, sensitive, specific, robust and automatable assay for nodavirus genotyping, making it a useful tool for diagnosis and screening for epidemiological studies.