We collected 56 clinical samples from the China Agricultural University Veterinary Teaching Hospital, including 25 canine-origin samples from dogs suspected of having canine parvovirus infection, 25 feline-origin samples from cats suspected of having feline parvovirus infection, and one confirmed positive sample each for canine distemper virus, canine coronavirus, canine parainfluenza virus, feline coronavirus, feline calicivirus, and feline herpesvirus. Detailed information is provided in attachment. According to the instructions provided with the Taq Pro U + Multiple Probe q-PCR Mix (Nanjing Vazyme Biotech Co., Ltd), the q-PCR reaction system was prepared as follows: 10 μL of 2 × Taq Pro U + Multiple Probe q-PCR Mix, 1 μM each of forward and reverse primers, 250 nM TaqMan Probe Mix, 0.4 μL of 50 × ROX Reference Dye 2, and 1 μL of template DNA. The remaining volume was adjusted with DNase/RNase-free deionized water to a final volume of 20 μL. The results obtained from this setup were used as the standard reference results. In addition, each sample was subjected to DNA sequencing (Sangon Biotech) to confirm whether the pathogen was CPV-2 or FPV.

A total of 50 VP2 gene sequences were obtained from the GenBank database, comprising 10 representative sequences from each of the following variants: CPV-2, CPV-2a, CPV-2b, CPV-2c, and FPV,¹ and sequence alignment was performed using Snapgene 6.1.1. Conserved regions were selected as target gene sites for the RPA-CRISPR/Cas13a system nucleic acid detection. The VP2 gene figments of CPV-2 and FPV were synthesized and cloned into the pUC57-Kan plasmid by Sangon Biotech (Shanghai) Co., Ltd. to serve as positive controls and quantitative standards (Supplementary Table S1).

The pCold-TF-Ulp1-SUMO-LwCas13a expression plasmid (Cas13a derived from Leptotrichia wadei), previously constructed in our laboratory, was transformed into E. coli BL21 (DE3) competent cells. Protein expression was induced with 500 μM IPTG at 18 °C for 18 h. Harvested cells were lysed using high-pressure homogenization, and the clarified lysate was filtered and purified via Ni-affinity chromatography using a HisTrap HP column (Cytiva lifesciences). Eluted fractions were analyzed by SDS-PAGE, and the target protein was further purified by cation-exchange chromatography. Final buffer exchange to storage buffer (500 mM NaCl, 50 mM Tris–HCl, pH 7.5, 5% glycerol, 2 mM DTT) was performed using centrifugal ultrafiltration. Protein concentration was measured with a BCA assay, aliquoted (50 μL), snap-frozen in liquid nitrogen, and stored at −80 °C.

Based on the alignment results in section 2.2, three crRNAs (crRNA-1, crRNA-2, and crRNA-3) targeting highly conserved regions of the VP2 gene were designed for the simultaneous detection of CPV-2 and FPV (Figure 1). When designing crRNAs to differentiate CPV-2 from FPV, previous studies have indicated that the central region of the crRNA-target RNA duplex, known as the “seed region” is sensitive to mismatches (Abudayyeh et al., 2016). We differentiated one region in the VP2 gene with two nucleotide mismatches between CPV-2 and FPV, and another region with a single mismatch, to which an additional artificial mismatch was introduced. As a result, three crRNAs with distinct mismatch patterns (crRNA-4, crRNA-5, and crRNA-6) were designed. Details of the mismatch sites are shown in Figure 1. The crRNAs were synthesized via in vitro transcription. Full-length crRNA primers containing the T7 promoter were annealed and used as templates for RNA transcription. The transcription was performed overnight at 37 °C using a T7 high-yield transcription kit (Nanjing Vazyme Biotech Co., Ltd). The resulting transcripts were purified using an RNA purification kit (Zymo Research), and the crRNA concentration was measured with a Nano-Drop 2000c (Thermo Fisher Scientific). The purified crRNAs were aliquoted and stored at −80 °C.

Based on the optimization experiments, two crRNAs with the highest detection efficiency were selected. Three pairs of RPA primers were designed in the conserved regions upstream and downstream of each crRNA target site. According to the orientation of the crRNA sequence, the T7 promoter was introduced into either the upstream or downstream primer to ensure that the transcribed target RNA after amplification would be complementary to the crRNA. The RNA reporter was labeled with FAM at the 5′ end and BHQ1 at the 3′ end. All RPA primers, crRNAs, and RNA reporter sequences were synthesized by GENEWIZ. The sequences used in this study are listed in Supplementary Table S2.

The RPA (Recombinase Polymerase Amplification) reaction was performed using the TwistAmp® Liquid Basic kit according to the manufacturer’s instructions with minor modifications. Each 12.5 μL RPA reaction contained the following components: 6.25 μL of 2 × Reaction Buffer, 0.9 μL of 25 mM dNTPs, 1.25 μL of 10 × E-mix, 0.6 μL each of forward and reverse RPA primers, 0.625 μL of 20 × Core Reaction Mix, 0.5 μL of DNA template, and 1.15 μL of DNase/RNase-free deionized water. Finally, 0.625 μL of MgOAc was added to initiate the reaction. The reaction mixture was incubated at 37 °C for 15 min to allow for amplification. After the RPA reaction, the amplification products need to be heated at 65 °C for 10 min to inactivate the proteins in the RPA system, allowing the proteins to separate from the amplified DNA and preventing the formation of smearing bands.

The CRISPR/Cas13a reaction system was established using target RNAs transcribed and purified from PCR-amplified pUC57-CPV and pUC57-FPV plasmids. For the detection of CPV-2 and FPV, the reaction mixture contained 45 nM Cas13a protein, 22.5 nM crRNA, 125 nM RNA reporter, 1 μL RNase inhibitor, 1.5 μL NEB r3.1 buffer, and 1 μL of target RNA. RNase-free ddH₂O was added to adjust the final volume to 20 μL. For differentiating between CPV-2 or FPV, the concentrations of key components were increased: Cas13a protein was used at 450 nM, crRNA at 225 nM, and ssRNA reporter at 1.25 μM. The remaining components and reaction volume were the same as described above. The mixture was incubated in a Real-time PCR System (Applied Biosystems) at 37 °C for 30 min, with fluorescence signals collected every 1 min.

A one-tube RPA-CRISPR/Cas13a detection system was developed using pUC57-CPV and pUC57-FPV plasmids as target DNA templates. For the detection of CPV-2 and FPV, the reaction mixture contained: 10 μL of 2 × Reaction Buffer, 25 mM dNTPs, 2 μL of 10 × E-mix, 0.48 μM forward primer, 0.48 μM each of forward and reverse primers, 20 × Core Reaction Mix, 0.5 μL T7 RNA Polymerase Mix, 0.5 μL RNase inhibitor, 25 mM rNTPs, 45 nM Cas13a protein, 22.5 nM crRNA, 125 nM ssRNA reporter, and 1.5 μL NEB Buffer r3.1, then, 1 μL of target DNA and 14 mM MgOAc were added to the tube cap (without mixing). The total volume was adjusted to 20 μL. After a brief centrifugation to combine all components, the reaction was incubated in a Real-time PCR System (Applied Biosystems) at 37 °C for 90 min, with fluorescence signals collected every 1 min. To further quantify the limit of detection, we analyzed the endpoint fluorescence signals of the detection system and evaluated the data from blank controls. The lower limit of detection (LLD) was defined as the lowest concentration at which the signal exceeded the mean of the blank control plus three standard deviations. RNase-free water was used as the template in the blank controls, and the values of X_blank and SD_blank were obtained from three independent replicates conducted using both the universal detection assay and the differential detection assay.

The endpoint fluorescence value output by the Real-time PCR System was used as the basis for optimizing the RPA-CRISPR/Cas13a fluorescence detection reaction. The optimization process included the selection of the most efficient crRNA and RPA primers. Additionally, key reaction parameters were systematically evaluated and optimized, including reaction time, temperature, Cas13a protein concentration, crRNA concentration, RNA reporter concentration, and the volume of RNase-free water used to dilute the reaction system.

The copy numbers of the constructed pUC57-CPV-2 and pUC57-FPV plasmids were calculated, and a 10-fold serial dilution was performed using RNase-free ddH₂O to achieve concentrations ranging from 10⁵ to 10¹ copies/μL. For each concentration, 1 μL of the diluted plasmid was used as the template in the RPA-CRISPR/Cas13a reaction. The endpoint fluorescence intensity was measured after 60 min of reaction to evaluate the sensitivity of the detection system. To assess the specificity of the RPA-CRISPR/Cas13a detection system, nucleic acids extracted from several other common pathogens of dogs and cats [including Canine distemper virus (CDV), Canine coronavirus (CCV), Canine parainfluenza virus (CPIV), Feline coronavirus (FCoV), Feline calicivirus (FCV), and Feline herpesvirus-1 (FHV-1)] were used as templates in the RPA-CRISPR/Cas13a reaction. Real-time fluorescence signals were recorded by Real-time PCR System (Applied Biosystems), and the endpoint fluorescence values were collected at 60 min to evaluate cross-reactivity and specificity.

Each reaction was performed in triplicate, and data were analyzed using GraphPad Prism version 10.3.1. A one-way ANOVA was conducted to evaluate the significance of differences among multiple groups for a single variable, while two-way ANOVA were used to compare differences between two groups. Data are presented as the mean ± standard deviation (SD). Asterisks (***) denote highly significant differences between groups (p < 0.001).

To establish a rapid and accurate method for the detection and differentiation of CPV-2 and FPV, we developed two one-tube RPA-CRISPR/Cas13a-based assays with high sensitivity and specificity. As shown in Figure 2, viral nucleic acids were extracted from clinical samples using an automated nucleic acid extraction system and served as templates for the RPA-CRISPR/Cas13a reaction. In the presence of target sequences, the products amplified by RPA and transcribed by T7 RNA polymerase are recognized by the Cas13a-crRNA complex, which activates the trans-cleavage activity of the Cas13a protein. This leads to the cleavage of a single-stranded RNA (ssRNA) reporter labeled with FAM at the 5′ end and BHQ1 at the 3′ end, generating a green fluorescence signal that can be visually observed under blue light or quantitatively measured using a fluorescence detector. The detection workflow first utilizes a universal detection system to determine whether the sample is infected with CPV-2 or FPV. If the result is positive, a differential detection system is then employed to distinguish between CPV-2 and FPV infections.

To identify optimal guide sequences for CRISPR/Cas13a-based detection, three candidate crRNAs (crRNA-1, crRNA-2, and crRNA-3) were designed and evaluated for the simultaneous detection of CPV-2 and FPV. All three crRNAs demonstrated robust activity, with fluorescence intensities significantly higher than the no-target control (NTC). Among them, crRNA-2 yielded the highest fluorescence signal at 30 min and reached peak intensity more rapidly than the other candidates, thereby being selected for subsequent universal detection assays (Figures 3A,B). Similarly, three additional crRNAs (crRNA-4, crRNA-5, and crRNA-6) were assessed for their ability to differentiate between CPV-2 and FPV. crRNA-4 produced strong fluorescence signals in the presence of both viral templates, whereas crRNA-6 failed to generate a signal significantly above that of the NTC, rendering it unsuitable for differential detection. Notably, crRNA-5 generated a strong fluorescence response with FPV but not with CPV-2, indicating its specificity for FPV. Therefore, crRNA-5 was selected for use in the differentiation assay between CPV-2 and FPV (Figures 3C,D).

To optimize the RPA reaction, three pairs of primers were designed for both the detection and differentiation systems, resulting in nine primer combinations tested for each. Using pUC57-CPV and pUC57-FPV plasmids as templates, RPA amplification was performed and evaluated by agarose gel electrophoresis to assess amplification efficiency. In the universal detection system, the primer pair RPA-F2/RPA-R3, targeting the crRNA-2 recognition region, exhibited the highest amplification efficiency and robust performance for both CPV-2 and FPV (Figures 3E,F), and was therefore selected for subsequent detection experiments. In the differentiation system, the RPA-F4/RPA-R5 pair also showed the highest amplification efficiency and strong performance for both CPV-2 and FPV (Figures 3G,H), and was chosen for further applications in distinguishing between CPV-2 and FPV.

To optimize detection performance, various reaction conditions were systematically evaluated based on the 30 min endpoint fluorescence intensity. For CPV-2 and FPV detection, the highest fluorescence signal was observed at a crRNA concentration of 45 nM; however, fluorescence intensities across different crRNA concentrations did not differ significantly. Similarly, a Cas13a concentration of 360 nM yielded the highest signal, though no statistically significant variation was observed among the tested concentrations. In contrast, fluorescence intensity increased proportionally with higher ssRNA reporter concentrations. To balance reagent usage with fluorescence visibility, the optimal conditions selected for subsequent detection assays were 22.5 nM crRNA, 45 nM Cas13a, and 375 nM ssRNA (Figures 4A–D). For differentiation between CPV-2 and FPV, the highest fluorescence intensity was obtained with 1,000 nM crRNA and 250 nM Cas13a. While increasing the ssRNA concentration initially enhanced fluorescence, the signal declined when ssRNA reached 1,000 nM. Accordingly, the optimized reaction conditions for the differentiation assay were set at 1000 nM crRNA, 50 nM Cas13a, and 875 nM ssRNA for subsequent experiments (Figures 4E–H).

To improve the performance of the one-tube RPA-CRISPR/Cas13a assay, key reaction parameters including time, temperature, and reagent composition were systematically optimized. Evaluation of fluorescence intensity under different conditions revealed that a 60-min incubation at 38 °C produced the most consistent and visually distinguishable results in both detection and differentiation assays (Figures 5A–C,E). We defined visibly detectable fluorescence by the naked eye as positive; however, this criterion may involve a certain degree of subjectivity. To further quantify the detection limit, we analyzed the endpoint fluorescence signals from two detection systems and tested the blank control data, which showed no significant differences among groups (Supplementary Table S3). Based on the mean and standard deviation of these blank controls, the limit of detection (LLD = Mean+3SD) was estimated using a standard formula to be approximately 120,000, therefore, for both detection systems, fluorescence values exceeding 120,000 at 60 min are uniformly considered positive. Although shorter reaction times (e.g., 30 min) also generated detectable signals, the fluorescence intensity at 60 min was much stronger, suggesting higher sensitivity. Extending the reaction beyond 60 min did not result in a substantial increase in signal, indicating that longer incubation times were unnecessary. Therefore, a 60-min reaction was selected to ensure assay robustness. Additionally, when combining the RPA amplification and CRISPR detection steps into a single-tube format, a marked decrease in sensitivity was observed, potentially due to enzyme interference affecting T7 transcription (Arizti-Sanz et al., 2020). Drawing on previous findings that dilution can mitigate such effects (Li et al., 2023), we tested the addition of RNase-free ddH₂O in varying volumes. A final addition of 20 μL RNase-free ddH₂O was found to enhance fluorescence signal without compromising reaction efficiency and was thus chosen for subsequent experiments (Figures 5D,H).

The copy number of the standard positive plasmid was calculated and serially diluted. Plasmid DNA ranging from (10⁵–10¹ copies/μL) was used as a template to evaluate the LOD of the RPA-CRISPR/Cas13a detection assay. For comparison, q-PCR was also performed using the same template concentrations. The results showed that RPA-CRISPR/Cas13a system achieved a detection LOD of 10² copies/μL for both CPV-2 and FPV VP2 genes (Figures 6C,D), which was slightly less sensitive than q-PCR, with a detection limit of 10¹ copies/μL (Figures 6A,B). In the CPV-2 and FPV differentiation assay, significant FPV-specific fluorescence was observed at template concentrations as low as 10³ copies /μL, while CPV-2 fluorescence remained comparable to the negative control across all concentrations (Figures 6E,F).

Specificity is a critical parameter for evaluating the performance of a diagnostic assay. To assess the specificity of the RPA-CRISPR/Cas13a-based detection system, nucleic acids from the following viruses were individually used as templates: canine distemper virus (CDV), canine coronavirus (CCV), canine parainfluenza virus (CPIV), feline coronavirus (FCoV), feline calicivirus (FCV), feline herpesvirus type 1 (FHV-1), CPV-2, and FPV. For the CPV-2 and FPV detection systems, only CPV-2 and FPV produced significantly elevated fluorescence signals, while the other six viruses generated signals comparable to the negative control (Figures 7A,B). Similarly, in the differentiation assay using crRNA-5 targeting the VP2 genes, only FPV produced a significantly higher fluorescence signal, whereas CPV-2 and all non-target viruses yielded minimal fluorescence (Figures 7C,D). These results demonstrate that the RPA-CRISPR/Cas13a-based assays possess high specificity for detecting and distinguishing CPV-2 and FPV.

To evaluate the clinical applicability of the RPA-CRISPR/Cas13a detection system, a total of 50 clinical samples were collected, including 25 canine-origin and 25 feline-origin samples. Nucleic acids extracted from these samples were tested using both q-PCR and the RPA-CRISPR/Cas13a assays. The detection results for the clinical samples are summarized in Table 1. According to the q-PCR results, 19 canine samples and 15 feline samples were positive, while the remaining samples were negative. The general RPA-CRISPR/Cas13a detection assay also identified 19 positive canine samples and 15 positive feline samples, fully consistent with the q-PCR results. Furthermore, the differential detection assay specifically identified all 15 FPV-positive samples, in agreement with the known origins of the samples. Overall, both the general and differential RPA-CRISPR/Cas13a assays demonstrated 100% concordance with q-PCR results, and were further validated by PCR and sequencing, indicating high accuracy and reliability for clinical diagnostics.