Positive samples for porcine rotavirus (PoRV), porcine circovirus II (PCV2), porcine reproductive and respiratory syndrome (PRRSV), pseudorabies virus (PRV), porcine parvovirus (PPV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), and Japanese encephalitis virus (JEV) are all stored in our laboratory. These samples include various types, such as tissue, fecal, serum, and samples from aborted fetuses. After being identified as positive, all samples are stored at −80°C. RNA or DNA was extracted from these samples using the TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China). cDNA was synthesized using PrimeScript RT Master Mix (Takara, Beijing, China), and both cDNA and DNA were stored at −80°C for future experiments.

To ensure the broad applicability and reliability of the detection method, the highly conserved VP6 gene was selected from the PoRV sequences of different strains for the construction of a standard plasmid. The sequence details can be found in Supplementary file 1. The RV-positive plasmid pMD18-RV-VP6 was then synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and validated by PCR. The target gene sequence can be found in Supplementary file 1, and primer details are provided in Table 1.

The pET28a-MH6-EsCas13d plasmid (Addgene, plasmid #108303, MA, USA) was transformed into Escherichia coli BL21 (DE3). The EsCas13d protein was then expressed and purified as previously described (Kushawah et al., 2020). To confirm the presence of the purified EsCas13d protein, we performed SDS-PAGE analysis. For this, we employed a mouse monoclonal anti-6 × His tag antibody (Sangon, Shanghai, China) as the primary antibody, and an HRP-conjugated rabbit anti-mouse IgG (Sangon, Shanghai, China) as the secondary antibody.

The PoRV VP6 gene was selected as the target for crRNA design using the CRISPR-DT tool (Zhu and Liang, 2019). The DNA template (gDNAs) is the reverse complement of the target crRNA sequence, synthesized with an additional T7 RNA polymerase promoter to facilitate T7 transcription, as detailed in Table 1. These gDNAs were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Using the T7 Quick High Yield RNA Transcription Kit (Beyotime, Shanghai, China), the gDNAs were transcribed into crRNA and purified using the Spin Column RNA Cleanup&Concentration Kit (Sangon, Shanghai, China). The concentration of crRNA was measured using the RNA Quick Quantification Kit (Thermo Scientific, MA, USA) and stored at −80°C for future experiments.

Based on previous studies (Li et al., 2020; You et al., 2024a), a buffer solution for CRISPR/EsCas13d trans-cleavage experiments was prepared, consisting of 40 mM HEPES (pH 7.1), 100 mM KCl, 10 mM MgCl₂, and 10% glycerol, referred to as Buffer 1. In this system, MgCl₂ primarily serves as a catalyst and plays a role in stabilizing the reaction environment. The total volume of the EsCas13d reaction system was 20 μL, comprising 10 μL Buffer 1, 500 nM EsCas13d, 200 nM crRNA, 10⁶ pM target RNA, 1 μL RNase inhibitor (2.5 U/μL), and 1 μM FQ-ssDNA reporter construct, with the remaining volume made up with nuclease-free water. The mixture was incubated at 37°C for 30 min in a QuantStudio™ 1PLUS real-time PCR system (Thermo Scientific, MA, USA), with fluorescence readings (FAM channl) collected every minute. After the reaction, the system was exposed to 365 nm UV light for visual inspection.

Primers targeting the PoRV VP6 gene were designed using Primer Premier 5.0 (Premier Biosoft, CA, USA). RPA amplification was performed using the RPA Nucleic Acid Amplification Kit (Warbio, Nanjing, China) according to the manufacturer’s instructions. The total reaction volume was 50 μL, containing 2 μL each of RPA-F and RPA-R primers (10 μM), 25 μL Buffer A, 13.5 μL ultrapure water, 2.5 μL Buffer B, and 5 μL template cDNA. Amplification was carried out at a constant temperature of 37°C for 30 min, and the products were analyzed using agarose gel electrophoresis.

The complete RPA-EsCas13d fluorescence detection system consisted of the following components: 500 nM purified EsCas13d, 200 nM crRNA, 1 μL T7 RNA polymerase (2.5 U/μL), rNTP mix (1 mM), 1 μL RNase inhibitor (2.5 U/μL), 1 μL template (amplified RPA product), 1 μM FQ-ssRNA reporter gene, 10 μL reaction buffer (Buffer 1), and nuclease-free water to bring the total volume to 20 μL. The mixture was incubated at 37°C for 30 min in a QuantStudio™ 1 PLUS real-time PCR system (Thermo Scientific, MA, USA), with fluorescence measurements (FAM) taken every minute. For the negative control (NC), 2 μL of nuclease-free water was used in place of the template.

The OP-RPA-EsCas13d detection platform integrates RPA amplification and Cas13d detection into a single reaction tube. The detection workflow is as shown by previous researchers (You et al., 2024b). RPA was performed at the bottom of the outer tube, with a 10 μL RPA reaction system consisting of 5 μL RPA Buffer A, 0.5 μL RPA Buffer B, 2 μM forward and reverse primers, 1 mM rNTP mix, 1 μL template cDNA (4 × 10⁶ copies/μL), and ultrapure water to complete the volume. In the inner tube, 20 μL of the EsCas13d reaction system was added, including 500 nM purified EsCas13d, 200 nM crRNA, 1 μM FQ-ssDNA reporter gene, 1 μL T7 RNA polymerase (2.5 U/μL), 1 μL RNase inhibitor (2.5 U/μL), and 10 μL Buffer 1. The tube was then sealed and placed in a thermal cycler or an isothermal module to ensure temperature stability. The RPA amplification was carried out at 37°C for 30 min. After amplification, the tube was briefly centrifuged to combine the EsCas13d system in the inner tube with the RPA product. The combined mixture was incubated at 37°C for an additional 30 min in the QuantStudio™ 1 PLUS real-time PCR system (Thermo Scientific, MA, USA), with fluorescence readings (FAM) collected every minute. For the negative control (NC), 2 μL of nuclease-free water was used in place of the template. The detailed flowchart of the detection process is presented in Figure 1.

The concentrations of ssDNA reporter molecules, EsCas13d, and crRNA are key factors affecting the efficiency of the CRISPR/EsCas13d reaction. The OP-RPA-EsCas13d method was optimized to enhance its performance. Additionally, the concentration of the ssRNA reporter gene was optimized across six levels (125, 250, 500, 750, 1,000, and 1,500 nM) to balance reagent cost and reaction performance. To address spatial effects and competitive interactions within the system, the ratio of Cas13d to crRNA was adjusted, using 200 nM crRNA and varying Cas13d concentrations from 100 to 600 nM. All experiments used nuclease-free water as a negative control.

To determine the concentration of the PoRV standard, the copy number was calculated using the corresponding formula. The plasmid was then serially diluted in 10-fold gradients using ddH₂O to obtain test samples with copy numbers ranging from 10⁷ to 10¹ copies/μL. Following the procedure outlined in step 2.8 and using the optimal reaction system, the test samples were analyzed using the OP-CRISPR/Cas13d detection system. After completion, the samples were observed under UV light to determine the detection limit of the OP-CRISPR/Cas13d system.

The performance of the OP-RPA-EsCas13d detection method was evaluated by testing 62 clinical sample. These samples included 40 fecal samples from diarrheic piglets and 22 intestinal tissue samples from pigs that died due to diarrhea. All samples were collected between April and October 2024 from vegetation-rich mountainous regions in southwestern China. For fecal samples, an appropriate amount of physiological saline is added, mixed thoroughly, and then centrifuged to collect the supernatant. For intestinal tissue, the tissue is homogenized in physiological saline, followed by centrifugation to obtain the supernatant. This process prepares the fecal and intestinal tissue samples. The OP-RPA-CRISPR/EsCas13d platform and RT-qPCR was used for detection. RT-qPCR primers and probes are shown in Table 1. The reaction mixtures, performed in three technical replicates, consisted of the following components: 10 μL of TaqMan™ Universal PCR Master Mix (Thermo Scientific, MA, USA), 200 nM of forward and reverse primers, 1 μL of probe, 2 μL of template cDNA, and the necessary amount of ddH2O to achieve a final reaction volume of 20 μL. The cycling conditions included an initial denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. In the RT-qPCR, a Ct value below 36 is considered positive.

Graphs were generated using GraphPad Prism 9.0 (GraphPad Software, CA, USA), and all data were presented as the mean ± standard deviation (SD) from three independent experiments. Statistical analyses were performed using SPSS 20.0 (IBM, Chicago, IL, USA). An unpaired t-test was used to compare data between two groups, while one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

We first developed a detection system using EsCas13d for the identification of PoRV. The EsCas13d protein was prepared according to previous methods. To demonstrate the cleavage activity of EsCas13d, eight reaction systems were selected for evaluation based on prior articles (Huang et al., 2023). The eight reaction systems each lack one to three essential components of CRISPR/EsCas13d. The results showed that significant fluorescence was observed only in Reaction 1, which included the target gene, FQ-ssRNA reporter protein, crRNA, and EsCas13d protein. This fluorescence could be detected using a quantitative fluorescence PCR instrument or observed with the naked eye under 365 nm UV light (Figure 2A). These results indicated that the purified EsCas13d protein we expressed had RNA cleavage activity. To further assess the specificity of the EsCas13d detection system, we tested multiple viruses, including PoRV, PCV2, PRRSV, PRV, PPV, CSFV, PEDV, and JEV. The results indicated that a strong fluorescence signal was produced only in the presence of PoRV, demonstrating its high specificity (Figure 2B).

To enhance the sensitivity of the entire RPA-CRISPR/EsCas13d system, we conducted screening and optimization of various key components and reaction conditions. Firstly, two pairs of PRA primers were designed based on the conserved region of the PoRV VP6 gene. The primers were tested in different combinations using RPA and gel electrophoresis to determine the optimal pair. Results showed that all primer combinations generated target bands of the expected size, while no bands were observed in the negative control due to the absence of RPA primers (Figure 3A). The R2/F2 primer combination exhibited relatively high amplification efficiency and minimal nonspecific amplification, with a product size of approximately 250 bp. Therefore, it was selected for subsequent experiments. For the detection of Porcine Rotavirus (PoRV), we designed three candidate crRNAs based on the conserved region of the PoRV VP6 gene. We then combined the RPA method with the EsCas13d cleavage system. Initially, nucleic acids were pre-amplified using RPA, and the resulting products were used as templates for detection by EsCas13d. Fluorescence signals were measured using a real-time PCR instrument. The results demonstrated that all three crRNAs effectively activated the trans-cleavage activity of EsCas13d, resulting in strong fluorescence signals (Figure 3B). Among them, crRNA2 exhibited the highest fluorescence intensity and cleavage efficiency, making it the preferred choice for further studies. To optimize the CRISPR/ESCas13d detection system, we systematically evaluated its components. Subsequently, we adjusted the ratio of Cas13d to crRNA, with a 1:1 ratio yielding the optimal fluorescence signal (Figure 3C). Finally, we optimized the concentration of the ssRNA reporter gene to balance reagent cost and performance (Figure 3D). The final reaction system for EsCas13d consisted of 10 μL buffer, 1 μM FQ-ssRNA, 200 nM crRNA, 200 nM EsCas13d, 10⁻⁶ pM target RNA, and 1 μL RNase inhibitor (2.5 U/μL), with the remaining components diluted in nuclease-free water.

Using the optimal reaction system determined in section 3.3, the sensitivity of the detection system was tested. We performed a tenfold serial dilution of the standard plasmid, with a dilution range from 1 × 10⁷ to 1 × 10¹, as shown in Figure 4. As the template concentration decreased, the detected fluorescence intensity also decreased; however, even at as low as 10 copies, a faint fluorescence was still emitted, classified as weak positive. This indicates that the detection limit of the CRISPR/EsCas13d detection system is 10 copies/μL.

To evaluate the feasibility of the RPA-EsCas13d detection system for identifying PoRV in clinical specimens, we analyzed 62 diarrhea samples from the Sichuan region of China. The samples included 40 fecal specimens and 20 intestinal tissue samples. Detection was performed using visible fluorescence, with a reaction time set to 30 min. The results showed that 5 out of the 62 clinical samples (coded as 6, 20, 26, 47, and 50) exhibited strong fluorescence signals (Figure 5), and the results were consistent with those obtained from RT-qPCR (Figure 5). The positive detection rate was 8.06% (5/62), confirming the consistency and reliability of the CRISPR/EsCas13d and RT-qPCR methods. For detailed information regarding RT-qPCR, please refer to Supplementary file 2.