PRV, PEDV and TGEV were donated by the Key Laboratory of Tropical Subtropical Animal Viral Diseases (KLATSAVD). PRRSV, PCV2, CSFV and PPV positive samples (confirmed positive by laboratory testing and sequencing) were retained by our laboratory.

Clinical blood samples (n = 30) were collected from pigs with suspected PRV infections across 3 farms in Yunnan Province, China, during 2023–2024.

The RAA Basic Nucleic Acid Amplification Kit enables rapid isothermal amplification of target DNA at 37 °C, while its companion RAA Basic Nucleic Acid Amplification Kit (Colloidal Gold) facilitates visual detection of amplified products—both kits were procured from AmpFuture Biotechnology Co., Ltd. And the CRISPR-Cas12a-specific nucleic acid detection strips (BaoYing TongHui Biotechnology Co., Ltd., Beijing, China) were employed for visual interpretation of nucleic acid detection results following CRISPR-Cas12a system activation. The gE sequence of porcine pseudorabies strain (PRV-XD-F3) was compared with the PRV gE sequences of GenBank numbered OR161244.1, OR161238.1, OR161226.1, OR161208.1, ON261936.1, and OR161232.1 in NCBI, and the PRV gE sequences were designed by using primer5.0 software. The primers and crRNAs were synthesized by General Biological Co.

The PRV gE gene PCR products were purified using the Universal DNA Purification Kit (Tiangen Biotech) following manufacturer’s instructions. Briefly, target bands were excised from 1% agarose gels, dissolved in B2 buffer, and purified through column adsorption. After washing steps, DNA was eluted in 15–40 μL buffer and stored at −20 °C for downstream applications.

The DNA product obtained was ligated into the pMD18-T vector. The ligation product was transformed into DH5a receptor cells, added to 890 mL of SOC medium and incubated for 60 min at 37 °C with shaking and cultured on L-agar medium containing X-Gal, IPTG and Amp. After colony growth, white colonies were selected with a small pipette tip and added to LB medium and incubated overnight on a shaker.

The bacterial solution was cultured overnight, DNA was extracted, and the PCR amplification system and procedure (target fragment amplification, recovery, and purification) was used. The PCR products were detected by 1% agarose gel electrophoresis, and the size of the bands was observed to determine whether the transformation was successful or not.

Recombinant plasmids were extracted using the Plasmid Extraction Kit following the manufacturer’s protocol. Briefly, bacterial cultures were pelleted, lysed with Buffers P1, P2, and P3, and centrifuged to remove debris. The supernatant was purified through an adsorption column, washed, and eluted in 50–100 μL elution buffer. Plasmid concentration was determined spectrophotometrically, and samples were sent for sequencing (Sangon Biotech (Shanghai) Co., Ltd.).

The concentration of validated recombinant plasmid was determined using a nucleic acid concentration meter. The average molecular weight of each base is 660. The pMD18-T-PRV-gE positive plasmid contains 4,500 bases, and the molecular weight of a single plasmid is 660 × (pMD18-T vector length + insertion gene length), and the final copy number is calculated according to the following formula (Kong, 2021).

Referring to the selection requirements of Cas12a protein PAM and protospacer sequences, ssDNA sequences were analyzed and crRNAs were designed using the CRISPR-DT online website,¹ i.e., TTTN sequences were identified within the highly conserved gE target region of PRV, and 23 nt was selected as the 23 bp target sequence.

Based on the determination and screening of crRNA recognition sequences, RAA primers were designed in their upstream and downstream regions by applying Primer5.0 software, respectively. Primer lengths were optimized for maximal amplification efficiency under the selected RAA conditions while maintaining target specificity.

The complete primer sequences and characterization are shown in Table 1.

Enzyme-free water was used as a negative control, and 1 μL Cas12a Naclease (1 μM), 2 μL pMD18-T-PRV-gE, 1 μL crRNA (1 μM), 1 μL F-Q (10 μM), and 2 μL of 10 × Cas12 reaction Buffer were added to a fluorescence octuple tube, and replenished to 20 μL with enzyme-free water. Next, the quantitative PCR was performed in a PCR instrument at 37 °C for 25 min, and the fluorescence signals were observed after passing through a UV lamp and a blue light.

The reaction temperature was set at 37 °C to meet the requirements for RAA amplification and to maintain optimal Cas12a cleavage activity. This was verified by a preliminary experiment.

The amplification system was constructed according to the RAA isothermal amplification kit: 25 μL of bufferA, 2.5 μL of bufferB (MgOAC), 2 μL of RAA upstream primer (10 μM), 2 μL of RAA downstream primer (10 μM), 16.5 μL of enzyme-free water, and 2 μL of target DNA, for a total of 50 μL of system. The reaction system was incubated on a metal bath at 37 °C for 20 min to complete the amplification of target DNA. After the reaction, an equal volume of nucleic acid extraction reagent (saturated phenol: chloroform: isoamyl alcohol = 25:24:1) was added to the RAA amplification product; after mixing, the reaction was centrifuged at 12,000 × g for 1 min; 7 μL of the supernatant was sucked up and detected by 1.5% agar gel electrophoresis to screen for the RAA primers.

PCR primers were established in our laboratory, the primer sequence: 5′-CATCTGGCTCTCTGCGTGCTGT-3′, 5′-TTGGGTCCATTCGTCACTTCC-3′, PCR amplification was performed using the pMD18-T-PRV-gE positive plasmid as a template with 10-fold isocratic dilution, PCR amplification system (25 μL): 2 × Taq PCR Master Mix 12.5 μL, 1 μL of each specific primer upstream and downstream, 1 μL of each reverse transcription product, 9.5 μL of sterilized double-distilled water, 1 μL of each reverse transcription product. PCR amplification system (25 μL): 2 × Taq PCR Master Mix 12.5 μL, 1 μL of each specific primer upstream and downstream, 1 μL of each reverse transcription product, and 9.5 μL of sterilized double-distilled water to make up the reaction system. The reaction system was supplemented with 9.5 μL of sterilized double-distilled water. The reaction system was placed on a fluorescence quantitative PCR instrument and pre-denatured at 98 °C for 10 s, followed by 55 °C for 30 s, 72 °C for 2 min, and then cycling the reaction for 35 times, and then detected by 1.5% agar gel electrophoresis.

Data were analyzed using GraphPad Prism 9.0. Fluorescence intensity differences were assessed via one-way ANOVA with Tukey’s post-hoc test. p < 0.05 was considered significant.

Using PRV nucleic acid as a template, the constructed recombinant plasmid standard pMD18-T-PRV-gE were identified by PCR, 1,868 bp had a specific target band appearing, which was consistent with the expected fragment size, and the negative did not show a band (Figure 2A). The sequencing results were compared with the sequence of gE of PRV-XD-F3 strain without base mutation or deletion, which proved the successful construction of pMD18-T-PRV-gE recombinant positive plasmid (Figure 2B-C). The standard positive plasmid was calculated as 4 × 10¹⁰ copies/μL according to the formula.

Based on the conserved region of the PRV gE gene, multiple sequence alignment was performed using Snap Gene software. Four crRNAs (crRNA1-4) targeting distinct PAM sites were designed via the CRISPR-DT online platform (see text footnote 1). To evaluate the trans-cleavage activity of crRNA-guided Cas12a, CRISPR/Cas12a detection systems containing individual crRNAs were constructed. Under excitation with ultraviolet (365 nm) and blue light (470 nm), all four crRNAs triggered cleavage of the fluorescent reporter molecule (FAM-TTATT-BHQ1), generating visible fluorescence signals (Figure 3A). Quantitative analysis of fluorescence intensity from blue light-transmitted images was performed Quantitative analysis of fluorescence signals demonstrated significantly higher trans-cleavage activity for crRNA1 and crRNA2 compared to crRNA3 and crRNA4 (p < 0.05), with no statistically significant difference observed between crRNA1 and crRNA2 (Figure 3B). For the PAM targets of crRNA1 and crRNA2, two pairs of RAA primers (RAA-F1/R1, F2/R2, F3/R3, F4/R4) were designed. All primer pairs specifically amplified target fragments. Notably, the RAA-F3/R3 primers paired with crRNA2 exhibited significantly higher amplification efficiency compared to other primers, with no detectable non-specific bands (Figure 3C). Based on these findings, crRNA2 and its corresponding RAA-F3/R3 primer pair were selected for subsequent experiments.

In order to optimise the CRISPR/Cas12a fluorescence assay, a screening procedure was performed for the concentration of Cas12a nuclease and crRNA. The experimental results demonstrated that the CRISPR/Cas12a detection system exhibited optimal target recognition and the strongest fluorescence signal when using 1.0 μL of Cas12a protein (1 μM) and 2.0 μL of crRNA (1 μM), corresponding to a crRNA:Cas12a molar ratio of 2:1 (Figure 3D-E).

The RAA-CRISPR/Cas12a detection system was established by optimizing the reaction components based on the manufacturer’s protocol.

Fluorescence signals were observed using probe F-Q under UV light (365 nm) and blue light (470 nm). Positive samples exhibited strong fluorescence, whereas negative controls showed no fluorescence (Figures 4A,B). A statistically significant difference was observed between the test system and negative control groups (^****p < 0.0001). For probe F-B, positive samples displayed distinct bands at both the quality control (C) and detection (T) lines, while negative controls (nuclease-free water) showed only the C line (Figure 4C). These results aligned with the expected outcomes.

To evaluate the specificity of the method, seven common porcine viral nucleic acids (PRV, PEDV, TGEV, PRRSV, PCV2, CSFV, and PPV) along with nuclease-free water were tested as negative controls. Fluorescence analysis revealed significant signals exclusively in the PRV-positive group (Figures 5A,B). Lateral flow strip results showed that only PRV samples generated both control (C) and test (T) lines, while other viral nucleic acids (PEDV, TGEV, PRRSV, PCV2, CSFV, PPV) and the negative control displayed only the C line (Figure 5C). These findings demonstrate that the RAA-CRISPR/Cas12a system exhibits high specificity without cross-reactivity with the genomic DNA of the other six pathogens tested.

To evaluate the sensitivity of the RAA-CRISPR/Cas12a system for PRV detection, a 10-fold serially diluted standard plasmid (1 × 10⁶ to 1 × 10⁰ copies/μL) was analyzed alongside nuclease-free water as the negative control. Fluorescence signals under blue light illumination were clearly detectable at a template concentration of 10 copies/μL (Figure 6A). Statistical analysis confirmed significant differences in fluorescence intensity between 10 copies/μL samples and negative controls (^****p < 0.001, Figure 6B), establishing the detection limit of the RAA-CRISPR/Cas12a fluorescence system at 10 copies/μL. Lateral flow strip analysis revealed distinct test line (T) bands for templates ranging from 10⁶ to 10² copies/μL. Faint but discernible T-line bands were observed at 10 copies/μL, while no bands appeared in negative controls (Figure 6C). In contrast, conventional PCR exhibited a detection limit of 1 × 10⁴ copies/μL, producing expected 354 bp amplicons only above this threshold (Figure 6D). These data demonstrate that the RAA-CRISPR/Cas12a system achieves 1,000-fold greater sensitivity than conventional PCR, confirming its high analytical performance for PRV detection.

A total of 30 blood samples were collected from clinical samples of a breeding farm in Yunnan Province, and nucleic acid extraction was performed, and RAA-CRISPR/Cas12a fluorescence, test strip method and PCR were compared with nucleic acid as the detection template. All three detection methods demonstrated identical positive rates of 36.7% (11/30) across the 30 clinical samples: conventional PCR (Figure 7A), RAA-CRISPR/Cas12a fluorescence assay (Figure 7B), and lateral flow strip analysis (Figure 7C). Comparative validation revealed 100% concordance between both CRISPR-based methods and PCR in sensitivity and specificity metrics. The results showed that the RAA-CRISPR/Cas12a fluorescence method, the test strip method, and all of the RAA-CRISPR/Cas12a fluorescence methods established in this study can be applied to the rapid clinical testing.