Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens

Introduction

Tilapia (Oreochromis spp.) is one of the most widely farmed freshwater fish species globally due to its high adaptability, fast growth, and excellent meat quality. Global production is estimated at 6,100,719 tonnes in 2020 (FAO, 2024). By providing sustenance, employment opportunities, and domestic and export revenues, this valuable species supports large populations worldwide (Wang & Lu, 2016). In the past decade, tilapia production has almost doubled (FAO, 2024), attributed mainly to its ease of cultivation, market demand, and stable pricing (Wang & Lu, 2016).

However, the production of tilapia has been threatened by several viral and bacterial diseases that can cause significant economic losses (Debnath et al., 2023; Soto et al., 2025; Senapin et al., 2025). Among the most important tilapia pathogens are tilapia lake virus (TiLV), infectious spleen and kidney necrosis virus (ISKNV), Francisella orientalis (FNO), and Streptococcus agalactiae (SAG) also called group B streptococcus (GBS) (Kawasaki et al., 2018; Machimbirike et al., 2019; Mabrok et al., 2021; Haenen et al., 2023; Alathari et al., 2023). Co-infections with these and other pathogens can exacerbate health and production issues in tilapia (Xu & Shoemaker, 2025). For example, from a 2015 to 2016 longitudinal study, SAG was the main bacterium detected in Nile tilapia farmed in a Brazilian reservoir and was mostly found concurrently with one or two bacteria, such as FNO and Aeromonas hydrophila (Delphino et al., 2019). Similarly, ISKNV has been detected in co-infections with SAG and other bacteria during high mortality events in Thailand (Dong et al., 2015) and Ghana (Ramírez-Paredes et al., 2021). Concurrent infections of TiLV and Aeromonas spp. have also been reported in mortality outbreaks in Egypt (Nicholson et al., 2017) and Thailand (Nicholson et al., 2020). Notably, mass mortality events of red hybrid tilapia have confirmed the presence of TiLV, SAG, and A. hydrophila, with mortality rates reaching up to 70% (Basri et al., 2020). Effective diagnosis and monitoring of these pathogens are crucial for disease management and control (Dong et al., 2023). Traditional diagnostic techniques such as virus and bacterial isolation, histopathology, immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA) are time-consuming, labor-intensive, and require specialized equipment and expertise (Dong et al., 2023). In contrast, molecular methods such as polymerase chain reaction (PCR) and quantitative PCR (qPCR) have gained widespread use in pathogen detection due to their high sensitivity and specificity, and rapid turnaround times (Soto et al., 2010; Kralik & Ricchi, 2017; Liamnimitr et al., 2018; Waiyamitra et al., 2018; López-Porras et al., 2019; Ramírez-Paredes et al., 2021; Taengphu et al., 2022; Dong et al., 2023).

During disease outbreaks, affected fish often harbor multiple pathogens (Dong et al., 2015; Delphino et al., 2019; Abdel-Latif et al., 2020; Basri et al., 2020; Ramírez-Paredes et al., 2021). Traditional diagnostic methods such as PCR and qPCR typically require separate tests for each pathogen, making the process time-intensive and costly, particularly when identifying multiple pathogens. In contrast, multiplex PCR allows for the simultaneous detection of multiple pathogens in a single sample by using multiple sets of primers, each specifically designed to bind to a specific DNA target. Although the sensitivity of multiplex PCR is generally lower than that of single-pathogen PCR or qPCR (Elnifro et al., 2000), it remains a practical and efficient diagnostic method, especially for clinical samples where pathogen loads are often high. Although multiplex PCR is efficient, the use of multiple primer sets in a single reaction can occasionally result in non-specific amplification. These challenges can be mitigated through careful primer design and optimization (Henegariu et al., 1997), underscoring the importance of sequence-based verification for accurate pathogen identification.

Sanger sequencing, a widely used DNA sequencing method, has limitations that restrict its use in detecting pathogens effectively in complex biological samples. It is relatively low throughput, sequencing one DNA fragment at a time, and requires a relatively high amount of pure DNA fragments (1 µg). Moreover, prior knowledge of the target region is essential for primer design, making it unsuitable for identifying novel or unexpected pathogens. Its reliance on specialized laboratory equipment and infrastructure further limits its portability, rendering it impractical for field-based diagnostics or use in resource-limited settings.

In contrast, Oxford Nanopore Technology (ONT) addresses these limitations by enabling on-site amplicon sequencing and offering several advantages for accurate pathogen detection (Delamare-Deboutteville et al., 2021). ONT requires only 50 ng of DNA per sample and can simultaneously sequence multiple amplicons of varying sizes, particularly short fragments (∼200 bp). This capability provides high sensitivity and precision for strain-level identification and differentiation—critical for understanding pathogen diversity, epidemiology, and tailoring disease management strategies. Its portability, scalability, and real-time sequencing capabilities make it especially suitable for field-based diagnostics and resource-limited settings, allowing rapid and reliable pathogen identification while avoiding delays associated with sample transport to distant centralized laboratories. By confirming target identity and resolving closely related strains, species, or novel variants, Nanopore sequencing complements PCR, offering insights beyond what PCR alone can achieve. Consequently, integrating multiplex PCR with ONT-based genotyping represents a powerful approach for aquaculture, enabling precise pathogen detection and characterization to enhance biosecurity and disease management.

We present a multiplex PCR assay with a detection level suitable for simultaneously identifying TiLV, ISKNV, F. orientalis, and S. agalactiae in sick tilapia samples. Using the portable Nanopore sequencing platform, we confirmed the presence of these pathogens at the sequence level and gathered genetic information from the amplicons. This assay offers a rapid and practical solution for detecting and genetically characterizing these pathogens, with the potential to complement existing diagnostic tools and inform targeted surveillance and control strategies.

Materials & Methods

Plasmid positive controls and the analytical sensitivity assay

All plasmid-positive controls except for S. agalactiae (constructed in this study) were obtained from our previous studies (Table S2). Positive plasmid control containing S. agalactiae groEL partial gene was obtained by cloning a 351 bp-groEL amplified fragment purified before being ligated into pGEM-T easy vector (Promega). A recombinant plasmid was subjected to DNA sequencing (Macrogen). The copy number of each plasmid was calculated based on its size in base pair (bp) and amount in nanogram (ng) using a web tool at http://sciprim.com/html/copyNumb.v2.0.html. A combination of four recombinant plasmids was mixed for the multiplex PCR sensitivity assay. This mixture was then subjected to 10-fold serial dilution, resulting in a 1 to 10⁶ copies/µl range. Subsequently, 4 µl of the diluted series was used in the multiplex PCR reaction. To simulate a clinical sample from a fish, each PCR reaction was spiked with 50 ng of RNA extracted from a healthy tilapia.

Results

Multiplex PCR detection of four pathogens among clinical samples

We have successfully optimized a multiplex PCR assay to simultaneously detect four important tilapia pathogens, TiLV, ISKNV, F. orientalis, and S. agalactiae, in a single reaction. The detection sensitivity of the new assay in the presence of spiked host RNA was 10³ copies/reaction for TiLV, ISKNV, and S. agalactiae. In the presence of 10⁴ copies of each template, the assay could detect F. orientalis and three other pathogens (Fig. S2). The assay could detect each pathogen efficiently in tilapia clinical samples, as confirmed by distinct PCR bands (Fig. 2). Some suspected co-infections detected by PCR were confirmed using Nanopore sequencing. Among the 15 clinical samples analyzed, nine exhibited co-infections involving three to four pathogens (Fig. 3). The assay was able to detect TiLV in heavily infected samples (3–7) both on the gel and by Nanopore sequencing but failed to detect it on the gel for samples (8–12) with low levels of TiLV infection. Similarly, the highest number of reads for ISKNV were found in sample #25 (twice more than in sample #27), yet it had a much fainter band on the gel (Table 1). There is a good correlation between read numbers and bands intensity for F. orientalis in four samples (#30–33) but only a weak band for S. agalactiae in one sample (#39) compared to number of amplicons (Figs. 2 and 3, Table S5). In some samples, we detected dual infections with more than one pathogen, as evidenced by the presence of multiple bands, including a possible co-infection with F. orientalis and TiLV in samples 3–7. However, differentiation of F. orientalis and ISKNV was challenging as their respective bands have similar sizes (Fig. 2).

Reads that failed stringent alignment to the pathogen gene panel were host-derived or partial pathogen sequences with lower nucleotide identity

Upon investigation of reads that failed to align, a significant proportion was found to map not only to the four pathogen gene segments but also to the host genome when a more lenient alignment configuration was employed (Fig. 4). Samples from run 2 (r2), predominantly derived from fish tissues, exhibited a higher percentage of reads mapping to the host genome than other samples. As expected, given the use of input PCR products as the template for all samples in run 1 (r1), no reads were found to align to the host genome when they were aligned with Minimap2 (default setting). On the contrary, a small portion of reads belonging to the host genome was found among different concentrations of single PCR products from run 3 (r3) (Fig. 4 and Table S5), which also consists of samples derived from tilapia organs (Table 1).

Discussion

The intensification of tilapia farming has led to a surge in emerging and re-emerging infectious diseases, resulting in substantial losses in the aquaculture industry (Machimbirike et al., 2019; Haenen et al., 2023; Shinn et al., 2023). In regions with high-density tilapia farming, diseases continue to evolve and intensify under strong selective pressures driven by climate change events, such as altered precipitation patterns, floodings, temperature fluctuations, and hypoxia (De Silva & Soto, 2009; Subasinghe et al., 2019). Inappropriate farming practices, such as the misuse of antibiotics, further exacerbate these challenges by contributing to the development of antimicrobial resistance (Kathleen et al., 2016; Santos & Ramos, 2018; Pepi & Focardi, 2021). Climate change also poses significant threats to food safety by altering pathogen growth rates and increasing the prevalence of parasites, bacteria, and viruses (Marcogliese, 2008; Short, Caminade & Thomas, 2017). Moreover, it amplifies risks to aquatic animal health by influencing pathogen virulence and host susceptibility (Barange et al., 2018). To address these climate-related risks, the implementation of effective, prevention-focused biosecurity plans is critical for sustainable aquaculture (FAO, 2018).

In this paper, we built upon our previous study (Delamare-Deboutteville et al., 2021), and improved the detection and genotyping of multiple pathogens by switching to multiplex PCR coupled with Nanopore sequencing. Our updated approach enabled the simultaneous and sequence-based detection and verification of four major tilapia pathogens: ISKNV, TiLV, S. agalactiae, and F. orientalis.

The need for simultaneous diagnosis of multiple pathogens is especially critical in addressing the high mortalities observed in larger grow-out tilapia. These mortalities are often exacerbated by co-infections, as highlighted by Ramírez-Paredes et al. (2021), whose research demonstrated that ISKNV-positive fish frequently harbor active co-infections with Streptococcus agalactiae and other bacterial pathogens. This underscores the importance of comprehensive diagnostic approaches to effectively manage and mitigate such complex disease scenarios.

Our primary goal in this study is to provide sequence-based confirmation and detect pathogen variants (some may potentially be more pathogenic, warranting different management strategies), rather than focusing on quantification. Sequencing directly from PCR products allows us to confirm the presence of target sequences and identify variants, even when using relatively universal primers, without the concern of false positives common with gel-based detection methods. While high Ct values offer increased sensitivity, they limit quantification due to primer concentration and depletion, reflecting the reaction endpoint rather than the original abundance. Accurate quantification is still best achieved through quantitative PCR (qPCR); however, the equipment is costly and rarely found in resource-limited lab settings. Furthermore, sequencing-based methods are compositional rather than absolute, even with DNA spike-ins, as they measure relative proportions. Our approach offers a complementary solution, providing reliable sequence-based confirmation and variant detection, filling a useful niche alongside traditional PCR methods.

Building on the complementary nature of sequencing-based methods, our multiplex PCR-Nanopore sequencing approach demonstrated greater sensitivity, detecting pathogen sequences in samples that tested negative by qPCR. However, these findings require cautious interpretation due to the limited sample size, necessitating further validation with larger cohorts. This method offers notable efficiency advantages, enabling simultaneous detection of multiple samples and targets of varying sizes in a single reaction, thereby reducing time and labor. Real-time sequencing with Nanopore technology provides immediate access to results as data is generated, unlike qPCR. DNA spike-ins at known concentrations can serve as positive controls, allowing for the estimation of relative abundance in positive samples and enabling operators to monitor and terminate sequencing runs once sufficient data is obtained. While the cost of sequencing reagents remains higher than that of qPCR, optimizing the number of samples and amplicon targets per library—through careful primer design while maintaining assay specificity and efficiency—can substantially reduce per-sample costs, making this approach increasingly competitive for high-throughput applications.

The discrepancies between gel-based interpretation of multiplex PCR products and qPCR or direct amplicon sequencing approaches are not surprising, given the subjective nature of gel visualization and the labor-intensive process of running multiple samples. The accuracy of pathogen detection can be significantly impacted by faint or similar-sized bands, making it challenging to standardize results. However, gel electrophoresis remains useful for validating Nanopore sequencing results. With high-degree sample-based multiplexing, it is now possible to perform high throughput sequence-based detection of multiple tilapia pathogens using Nanopore sequencing. The utilization of this application is paramount for the aquaculture industry, given that disease outbreaks are frequently linked with multiple infections (Abdel-Latif & Khafaga, 2020; Huang et al., 2020; Liu et al., 2020; Basri et al., 2020). Furthermore, as aquaculture farms are often situated in remote locations far from diagnostic reference laboratories, the ability to mobilize diagnostic testing with rapid results, high accuracy, and less complicated equipment near the farm is preferred.

Although our approach is scalable, there are limitations due to the old version of the operating system MinKNOW used during the study. Short amplicons are sequenced less efficiently and have lower read accuracy. We addressed this issue using a stringent read filtering and alignment approach, which led to a reduced read recovery in the final abundance calculation. The higher error rate of Nanopore reads has resulted in inconsistent outcomes when using different mapping alignment strategies, with more stringent parameters improving specificity at the cost of sensitivity, and vice versa. However, with recent advancements in Nanopore read accuracy, we anticipate this issue will diminish moving forward. These improvements are expected to enable both high sensitivity and high specificity with greater confidence, facilitating the reliable detection of pathogen and their novel variants. Some synthetic samples analyzed contained reads from other pathogens, which was unexpected and suggests cross-ligation of native barcodes during library preparation. The availability of unbound adapters is higher among samples with low DNA input, leading to higher crosstalk levels and reads from other samples. New improvements in the current Nanopore technology and library preparation protocols (as of 23 January 2025) could mitigate these issues. This includes (1) Switching to a PCR-barcoding kit to only enrich for amplicon with Nanopore partial adapter, (2) the use of a new Q20+ LSK114 kit with improved read accuracy, and (3) the use of high accuracy Dorado base-calling model (https://github.com/nanoporetech/dorado).

By enforcing a stringent alignment setting, the reads aligning to the pathogen gene segment are directly suitable for subsequent reference-based variant calling, producing highly accurate variants useful for biological interpretation. This is exemplified by the consistent recovery of a TiLV segment 9 variant previously only found in a Vietnamese TiLV strain in all Thai tilapia samples from the same sampling batch. Considering the proximity of Thailand and Vietnam, this may suggest a possible transboundary event, highlighting the need for more thorough sampling and sequencing of additional segments to elucidate the degree of TiLV diversity in both regions.

For field diagnostics, some of the equipment used in the present study can be replaced with portable items that are small enough to fit in a bag for use in remote settings. An example of the field application of this diagnostic workflow is the Lab-in-a-backpack concept developed by WorldFish (WorldFish, 2020; Huso et al., 2020; Cagua et al., 2021; The University of Queensland, 2021; Chadag et al., 2021; Barnes et al., 2021). It includes all the necessary sampling, extraction, and sequencing kits, pipettes, and consumables, as well as the use of the miniPCR thermocycler and blueGel electrophoretic system from miniPCR bio™, a minicentrifuge, a magnetic rack, a fluorometer, a Nanopore MinION sequencer, and flow cells for sequencing. Additionally, a laptop with minimum requirements to run the MinKNOW software is required. Similar PCR-sequencing approaches using portable equipment have been employed for the rapid identification of a diverse range of biological specimens, including plants, insects, reptiles (Pomerantz et al., 2022), and viruses such as COVID-19, Ebola, and Zika (González-González et al., 2019; González-González et al., 2020).

Improvements in the remote and low-resource applications of the current methodology will broaden its use for capacity building and implementations in low-income countries, where high mortalities due to unknown causes are prevalent in tilapia farming. Accurate and rapid genomic detection of fish pathogens through our amplicon-based approach will allow health professionals to promptly provide advice and take necessary actions for producers. Moreover, it can facilitate informed decisions regarding additional investigations, such as whole-genome sequencing (WGS), on isolates stored in biobanks. The sequence data obtained from WGS offer crucial epidemiological insights, which can be utilized to develop customized multivalent autogenous vaccines using local pathogens (Barnes et al., 2022). These vaccines can then be administered to broodstock and seeds before distribution among grow-out farmers for restocking purposes.

Conclusions

In summary, our study presents an attractive approach for detecting and verifying four tilapia pathogens in clinically sick fish, which can also be applied to pathogens in other livestock, fish, or crustaceans if the genetic information of the pathogen is publicly available for primer design and reference mapping. Although there are limitations to the current pipeline version used at the time of this study, we are optimistic that the current improvements in Nanopore technology will further enhance the accuracy and scalability of our approach.

Supplemental Information