The complete coding sequence of major capsid protein (MCP) gene from a reference strain LMBV-FS2021 (GenBank accession: ON418985.1) was selected for bioinformatics analyses. The eight pathogens, including Aeromonas veronii (A. veronii), Streptomyces xiamenensis (S. xiamenensis), Plesiomonas shigelloides (P. shigelloides), Citrobacter freundii (C. freundii), Micropterus salmoides rhabdovirus (MSRV), Red spotted grouper nervous necrosis virus (RGNNV), Snakehead vesiculovirus (SHVV) and Grass Carp Reovirus (GCRV), were used in the specificity assay.

In this study, a total of 42 clinical samples of fish liver (samples #1 to #42) with clinical characteristics were collected at a commercial fish farm in Nanyang, Henan Province, China. The primary clinical symptoms of the diseased fish exhibited ulcer lesions and mild bleeding appeared on the body surface. Among these samples, 19 samples tested positive and 23 samples tested negative for LMBV by qPCR (Novoprotein, China) (Supplementary Table S1).

The plasmid containing the full-length MCP gene of LMBV-FS2021 (GenBank accession: ON418985.1) (Supplementary Table S2) was kindly provided by Dr. Qin (Qin et al., 2024). MCP gene conservation was assessed across all 19 sequences obtained from GenBank using the query: “((LMBV[Title]) OR Largemouth Bass Virus[Title]) AND COMPLETE.” Phylogenetic analysis was performed using the neighbor-joining algorithm in MEGA X software. Multiple sequence alignment was generated using MUSCLE, and tree robustness was evaluated with 1,000 bootstrap replicates. Viral strains analyzed in this study, including their information and GenBank references are presented in Supplementary Table S3.

In the RPA procedure, a kit (GenDx Biotech, China) was employed following the report with slight adjustment (Huang et al., 2024). Briefly, the reaction solution was assembled in a final volume of 24 μL, comprising 10 μL of reaction buffer, 5.0 μL of ERA buffer, 1.5 μL of forward primer (10 μM), 1.5 μL of reverse primer (10 μM), 3.0 μL of template DNA (concentration adjusted according to specific applications), and 3.0 μL of ddH₂O. The mixture was vortexed briefly and centrifuged prior to the addition of 1.0 μL of activator (magnesium acetate, 200 mM). The reaction was then incubated at 39°C for 20 min. The RPA products were subsequently used for downstream applications.

Within a 20 μL CRISPR assay, the reaction solution of 15 μL (excluding the template) was assembled by combining the components as follows: 8.8 μL ddH₂O, 2 μL reaction buffer, 0.2 μL RNase inhibitor (Novoprotein, China), 1 μL FAM-ssDNA-BHQ1 reporter (Genewiz, China), 2 μL crRNA (1 μM, synthesized by Genscript, China), 1 μL engineered enAsCas12a-Ultra protein (enAsU-R). Subsequently, 5 μL RPA product was added into the CRISPR reaction tube. The mixture was then subjected to incubation at 37°C for 20 min. Following this, fluorescence signals were captured using an LED Transilluminator (Sangon, China). For accurate quantification of fluorescence, the cleavage results of the CRISPR reaction represented by the fluorescence intensity were obtained using a Varioskan^® LUX multimode microplate reader (Thermo Scientific, United States). The excitation wavelength was set to 485 nm, and fluorescence emission was collected at 535 nm (Huang et al., 2023; Huang et al., 2024).

The crRNA candidates of LMBV MCP gene (Table 1), each 42 nucleotides (nt) in length, were designed following the principles of crRNA design (motif of TTTN) and EasyDesign (Huang et al., 2024). The crRNA screening was performed using the DNA template of the MCP gene with a Cas12a-based method over a 60-min fluorescence recording period. The crRNA with the highest reactivity was selected for subsequent RPA primer screening. RPA primer pairs were designed following the guidelines provided by the TwistAmp^™ Basic Kit (TwistDx, United States) and the EasyDesign online design tool. The main design principles include the following: The GC content of the primers is between 35 and 60%; No more than 4 consecutive base forms of A, T, C, and G within the primer; No G within the 3 nt interval at the 5′ terminal of the primer; The primers contain G or C within the 3 nt interval at the 3′ terminal (Huang et al., 2024). The RPA primer candidates for optimal primer screening are listed in Table 2.

The LMBV DNA was detected by real-time qPCR assay. DNA was isolated from approximately 30 mg dry homogenized liver tissue. All DNA isolation was done using the RNA/DNA nucleic acid extraction kit (Bioer Technology, China) according to manufacturer’s protocol. The DNA concentration and purity were determined by NanoDrop ONE (Thermo Scientific, United States). Simply, the reaction system (NovoStart^® SYBR qPCR SuperMix Plus, Novoprotein, China) contained 2 × qPCR SYBR Green Master Mix (10.0 μL), 10 μM forward primer (0.4 μL), 10 μM reverse primer (0.4 μL), 50 × ROX Reference Dye 1 (0.4 μL), and template cDNA (2.0 μL), and ddH₂O was added to a total reaction volume of 20 μL. Then qPCR was performed on the CFX96 Duet Real-Time PCR system (Bio-Rad, United States). The following thermocycling conditions were used for qPCR: 3 min pre-incubation at 95°C, followed by 40 cycles of 95°C for 5 s; 60°C for 30 s. The threshold cycle (Ct) value of the samples was quantified by qPCR using a pair of MCP gene-specific primers. The target region and primer sequences for qPCR detection of LMBV MCP gene are provided in Supplementary Table S4. The samples were defined as positive if its Ct value was ≤33. Conversely, samples with a Ct value > 33 or undetectable Ct values were defined as negative.

In the specificity assay, 8 pathogens, including A. veronii, S. xiamenensis, P. shigelloides, C. freundii, MSRV, RGNNV, SHVV, and GCRV, were tested using the RPA-CRISPR/Cas12a method, all the DNA templates were normalized to 10⁸ copies per reaction. In the sensitivity assay of LMBV, a gradient dilution of the DNA templates was tested, including 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 50, 10, and 0 copies per reaction. The limit of detection for LMBV was determined based on the fluorescence signal obtained from the RPA-CRISPR/Cas12a detection.

Lateral flow strip assay (LFA) was carried out as previously described (Liu Y. et al., 2024) by using commercially available detection strips (GenDx Biotech, China). The CRISPR/Cas12a reaction system for LFA utilized biotin-ssDNA-FAM in place of FAM-ssDNA-BHQ1, while all other components remained unchanged. The strips were designed to detect amplicons dual-labeled with FAM and biotin, and the results were visually identified following the manufacturer’s instructions. For the assay, 20 μL of the Cas12a reaction mixture was added to a tube containing 80 μL of HybriDetect Assay buffer. The mixture was vortexed and centrifuged, after which a lateral flow dipstick was inserted and incubated at room temperature for 2 min. Following incubation, the dipstick was removed and imaged using a smartphone camera.

A total of 42 clinical samples were analyzed using the established RPA-CRISPR/Cas12a assay. As illustrated in Figure 1, the RPA-CRISPR/Cas12a assay for LMBV detection, involved the following steps: DNA was first extracted from fish-derived samples. The extracted DNA was then subjected to detection of LMBV using the RPA-CRISPR/Cas12a assay described above, employing the optimized crRNA and RPA primers. Results were obtained through three approaches: visual inspection, fluorescence quantification (using a microplate reader), and lateral flow strip analysis. The recombinant pET28-MCP plasmid served as the positive controls (Pos), while ddH₂O was used as the negative control (Neg). In the absence of the target nucleic acid sequence, only the control line (C) is visible, while the test line (T) remains invisible.

The one-pot RPA-CRISPR assay combines RPA with CRISPR/Cas12a detection in a single-tube reaction. The RPA reaction was prepared at the bottom of the tube, with the following components: 10 μL of RB reaction buffer, 4 μL ERA buffer, 1.25 μL each of forward and reverse (10 μM), 1 μL of DNA template, 1.0 μL of magnesium acetate (200 mM), and ddH₂O was added to a total final volume of 20.0 μL. The CRISPR/Cas12a detection, a 5 μL mixture was prepared in tube cap, containing 1 μL of enAsU-R Cas12a protein (200 ng/μL), 1 μL of crRNA (20 μM), 1 μL of FAM-ssDNA-BHQ1 reporter (1 μM), 0.1 μL of RNase inhibitor (40 U/μL), 1.9 μL of ddH₂O. The reaction was performed at 39°C for 10 min for RPA amplification. Following amplification, the CRISPR/Cas12a reaction system was manually mixed with the RPA amplicons by gentle tube inversion (5–10 times) and incubated at 39°C for 30 min.

The lyophilized reagents in the reaction tube for the RPA-CRISPR system include all components except the RPA activator (magnesium acetate) and the target DNA sample. Tubes were briefly centrifuged and stored at −80°C until ready for lyophilization overnight (FreeZone^® Fully Automatic Top-Loading Capping System, LABCONCO, United States). After lyophilization, the tubes are stored at −20°C or 4°C. For detection, the RPA activator, sample DNA, and ddH₂O are added directly to the reaction tube containing the lyophilized pellet. The mixture should be thoroughly mixed before incubation at 39°C for 40 min. Fluorescence signals were then captured using an LED Transilluminator (Sangon, China).

To ensure the precision and dependability of the experimental outcomes, each test was conducted in triplicate and subjected to statistical evaluation using GraphPad Prism 8.0. The results are presented as mean ± SEM, and an analysis of ANOVA was performed to assess the differences between experimental group and control group. Statistical significance was defined as a p value of <0.05.

To improve the detection of clinical LMBV infections, selecting highly active crRNA is essential for the Cas12a-based fluorescence assay. Phylogenetic analysis of available sequences revealed two major types of LMBV MCP genes (Figure 2A), type A and type B, differing by 12 nucleotide (nt) out of the 1,392-base segment. Based on these sequences, 5 crRNA candidates (cr1-cr5) targeting the conserved regions of LMBV MCP gene were designed (Table 1, Figures 2B–F), with the nt location of 90–114, 105–129, 120–144, 561–585, and 1,278–1,302 in the coding sequence of MCP gene, respectively.

During crRNA screening, template DNA representing LMBV MCP sequences (Supplementary Table S2) was tested in Cas12a based fluorescence activity assays with the five crRNAs. Among these, MCP-cr2 exhibited the highest fluorescence activity in the results of the microplate reader (Figures 2G,I) and the direct observation (Figure 2H), and then was selected as the target crRNA for subsequent experiments.

Using the optimal crRNA MCP-cr2 (Table 1), three pairs of RPA primers (Table 2) were designed and screened to optimize the pre-amplification efficiency of the MCP gene (Figure 3A). Each forward and reverse primer combination was evaluated for amplification performance. The results indicated that the primer pair F1-R1 exhibited the highest fluorescence intensity in all the primer pairs, making it the most efficient RPA primer pair for LMBV detection (Figures 3B–D). Thus, the primer pair F1-R1 was selected for the subsequent experiments.

To assess the sensitivity of the RPA-CRISPR/Cas12a fluorescence assay, MCP gene templates with concentrations of 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 50, 10, and 0 copies per reaction were tested. The results of naked-eye observations and the microplate reader demonstrated that the limit of detection (LOD) for the MCP gene was 50 copies per reaction (Figures 4A,B).

For the specificity assay, fluorescence activity was observed exclusively with the LMBV DNA template. In contrast, DNA templates from other organisms, including A. veronii, S. xiamenensis, P. shigelloides, C. freundii, MSRV, RGNNV, SHVV, and GCRV, exhibited fluorescence levels comparable to the negative control, indicating no significant fluorescence in these reactions (Figures 4C,D). Furthermore, a distinct test band was observed on the Lateral flow strips, confirming that the visual detection system possesses high specificity (Figure 4E).

For clinical nucleic acid detection of LMBV, the process outlined in Figure 5A was implemented. Sample collection is carried out on-site, either in the field or near breeding tanks. Following nucleic acid extraction, the presence or absence of the target nucleic acids is determined using the RPA-CRISPR/Cas12a fluorescence detection method. A total of 42 fish liver samples were tested. To comprehensively assess the feasibility of the established assay for detecting LMBV samples, RPA-CRISPR/Cas12a and the gold standard qPCR were used for comparative validation. Both methods were repeated 3 times, and the consistency of the 2 methods was mutually analyzed. Among these, qPCR analysis identified 19 positive samples and 23 negative samples (Supplementary Table S1). The results of RPA-CRISPR/Cas12a fluorescence (Figure 5B) assay demonstrated a 100% concordance rate with the results of qPCR (Figure 5C) and LFA (Figure 5D). Notably, samples with low Ct values in the qPCR assay exhibited high fluorescence signals in the RPA-CRISPR/Cas12a method, indicating strong agreement and consistent performance between the different detection methods.

To facilitate the rapid application of the RPA-CRISPR method for on-site detection, we attempted a one-pot approach for sample detection. The results from six samples (Figure 5E) demonstrated that the one-pot method effectively detected target nucleic acids, yielding results consistent with the conventional two-step method. To improve detection system stability for field applications, we explored lyophilization strategy. Figure 5F shows the reaction mixture’s transition from liquid state before lyophilization to a near-microsphere state after freeze-drying, which enables more stable storage of the detection system. Testing six samples using this lyophilized reagent confirmed that the method effectively detected the target nucleic acids, with results consistent with the two-step method (Figure 5G). These findings demonstrate that both the one-pot approach and lyophilization strategy facilitate rapid and convenient detection of LMBV.