Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3

Simple Summary Porcine circovirus type 3 (PCV3) is a newly discovered pathogen that causes porcine dermatitis and nephrotic syndrome (PDNS)-like clinical signs. The current lack of effective vaccines and therapies for PCV3 has led to a main reliance on early diagnosis and detection for the prevention and control of PCV3 infection. Nucleic acid detection is commonly used for the early identification of pathogens, and the sensitivity and specificity of two-pot RPA-CRISPR/Casa-Ps, the rapid, effective, sensitive, and specific detection method for PCV3 is, indeed, needed. Here, we develop one- and two-pot methods with a combination of recombinant polymerase amplification (RPA) and a CRISPR/Cas12a detection system for the simple, rapid, sensitive, specific, and visual detection of PCV3 clinical samples within 30 min. These two detection methods have the potential to detect clinical PCV3 infections. Abstract The porcine circovirus type 3 (PCV3) infection is an emerging disease associated with clinical signs of porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs. Currently, there is a lack of effective vaccines and therapeutics against this disease. Therefore, rapid, effective, sensitive, and specific detection methods are crucial for the timely identification, prevention, and control of PCV3. In this study, we developed one- and two-pot visual detection methods for PCV3 using a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a detection system combined with recombinase polymerase amplification (RPA). These two methods demonstrated no cross-reactivity with eight other swine viruses and exhibited minimum detection limits of five and two copies of viral DNA, respectively, revealing their high specificity and sensitivity. During a clinical sample detection within 30 min, the coincidence rates between the one- and two-pot detection methods and real-time quantitative polymerase chain reaction (qPCR) were 100%. In conclusion, both one- and two-pot RPA-CRISPR/Cas12a detection methods have significant potential for the rapid, sensitive, and specific visual detection of PCV3.


Introduction
Porcine circoviruses (PCVs), belonging to the Circovirus genus of the Circoviridae family, are small, non-enveloped viruses with single-stranded circular DNA genomes [1].
The spread of PCV3 has caused severe economic losses in the swine industry.To date, there are no commercial vaccines or effective drugs against this disease, which makes early detection and monitoring of pathogens particularly important for preventing and controlling PCV3 infection.Serological detection mainly depends on various enzymelinked immunosorbent assay (ELISA) formats owing to their high throughput and low cost [8,9].A limitation of these detection methods is that the samples need to be obtained a long time after viral infection, which misses the early detection period.In contrast, nucleic acid amplification tests, such as polymerase chain reaction (PCR) and reverse transcription (RT)-PCR, can provide the fast, sensitive, and specific detection of early viral infections [10,11].However, these detection methods commonly require specialized equipment, trained technicians, and a certain reaction time (approximately 2 h), which is not suitable for real-time detection of pathogens.
In this study, we developed one-and two-pot RPA-Cas12a for the visual detection of PCV3.The whole reaction of the one-and two-pot assays was completed within 30 min at 37 • C with a sensitivity of viral DNA detection under five and two copies, respectively.In addition, 25 clinical tissue samples were analyzed by both one-and two-pot RPA-Cas12a methods, in which the coincidence rate of test results with real-time quantitative PCR (qPCR) was 100%, indicating the reliability and applicability of these two methods in clinical PCV3 diagnosis.

The Expression and Purification of Cas12a Protein
A full-length fragment of Cas12a was amplified from the 6 His-MBP-TEV-huLbCas12a plasmid (#90096; Addgene, Beijing, China) and cloned into the pET-28a vector to generate the pET-28a-Cas12a expression plasmid.Following validation by sequencing, this plasmid was transformed into BL21 competent cells (DE3) (TSC-E06; Tsingke Biotech, Beijing, China) to express the Cas12a protein.Cas12a proteins were purified using a His-tag protein purification kit (DP101-01; Transgen Biotech Co., Ltd., Shenzhen, China) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.The purified Cas12a proteins were stored at −80 • C. The pcold-TF-PCV3 Cap plasmid (PCV3 Cap DNA) was maintained in our laboratory.

The Optimization of Recombinant Polymerase Amplification (RPA)
Three pairs of RPA primers targeting the PCV3 Cap gene were designed using Snapgene software v7.2.The RPA reaction system was set to a 50 µL final reaction volume, including 30 µL buffer, 2.5 µL each of forward and reverse primers (10 µM), 1 µL PCV3 Cap DNA, 2.5 µL Magnesium Acetate (MgOAC, 280 nM), and appropriate sterile water.The RPA reaction was performed at 37 • C for 20 min using the TwistAmp Basic Kit (TABAS03KIT; Twist D X, Cambridge, UK), followed by 1% agarose gel electrophoresis.

Establishment and Sensitivity of One-Pot RPA-CRISPR/Cas12a-PCV3 Detection Method
To simplify the procedure and reduce contamination, we adapted the RPA amplification step and the CRISPR/Cas12a-PCV3 detection step in one tube by adding the amplification reagents to the bottom of the tube and the detection reagents to the cap of the tube, followed by CRISPR/Cas12a detection.The total reaction system was set to 40 µL final reaction volume to optimize reaction sensitivity.The total reaction volume of 40 µL included 32 µL amplification reagent (15 µL buffer, 1.5 µL each of forward and reverse primers, 1 µL viral DNA of various concentrations, 1 µL MgOAC, and 12 µL sterile water) at the bottom of the tube and 8 µL CRISPR/Cas12a-PCV3 detection reagent (1 µL Cas12a, 1 µL crRNA, 2 µL ssDNA-FQ probe, 4 µL NEB Buffer).The one-tube RPA-CRISPR/Cas12a-PCV3 detection procedure as follows: after the RPA amplification at 37 • C for 20 min, the tube was centrifuged and then incubated at 37 • C for 10 min to perform CRISPR/Cas12a-PCV3 detection.

The Detection of Clinical Samples
DNA was extracted from 25 clinical samples (200 mg/sample) in 1 mL PBS through grinding, freeze-thaw, and centrifugation using a viral DNA purification kit and then detected by qPCR using the reported primers [21] and one-or two-pot RPA-CRISPR/Cas12a-PCV3 detection methods.All positive samples were further validated by sequencing.

Statistical Analysis
Significant differences were evaluated using one-way analysis of variance (ANOVA) or Student's t-test using Prism 8.0 software (GraphPad Software, Boston, MA, USA), with a p value < 0.05 considered statistically significant.

Expression and Purification of Cas12a Protein and Assay of Endonuclease Activity
The Cas12a gene was amplified from the 6 His-MBP-TEV-huLbCas12a plasmid and cloned into the pET28a vector (Figure S1A).To obtain Cas12a proteins, a prokaryotic expression system was used for expression.Cas12a proteins were mainly expressed in the bacterial lysate supernatants (Figure S1B), which were purified and detected by SDS-PAGE and Western blotting (Figure S1C,D).Subsequently, the endonuclease activity of the purified Cas12a protein was assayed by ultraviolet light, blue light, and fluorescence signals at 37 • C for 30 min in an RNase-free centrifuge tube containing detection buffer, the known-target DNA (KT-DNA) and crRNA (KT-crRNA) [21], and a synthesized ssDNA-FQ probe.The results showed that both blue and ultraviolet light were positive (Figure S1E), which was consistent with the fluorescence signals (Figure S1F), suggesting that the purified Cas12a protein had good endonuclease activity.

The Optimization of Cas12a and crRNA Concentration in CRISPR/Cas12a-PCV3 Detection Method
Three crRNAs targeting the PCV3 Cap gene were designed and synthesized by GE-NEWIZ, according to the PAM sequence (TTTN) recognized by Cas12a (Figure S2A, Table S1).To screen the best crRNA, three crRNAs were assayed in the CRISPR/Cas12a-PCV3 detection method at 37 • C for 30 min, respectively.The results showed that the fluorescence signal of crRNA3 was the strongest, and so it was selected for subsequent experiments (Figure S2B,C).Next, the concentrations of Cas12a and crRNA3 were optimized in the CRISPR/Cas12a reaction based on their fluorescence signals.The results showed that the appropriate concentrations of Cas12a protein and crRNA3 were 0.5 µg/µL and 0.5 µM, respectively (Figure S2D,E).

Establishment of Two-Pot RPA-CRISPR/Cas12a-PCV3 Detection Method
The two-pot RPA-CRISPR/Cas12a-PCV3 detection method was established by combining RPA amplification and CRISPR/Cas12a-PCV3 detection, with further optimization of the total reaction time.The amplification time of RPA was set to 20 min, according to the RPA amplification kit.Subsequently, the various times (5, 10, 20, 30, or 40 min) were optimized in the CRISPR/Cas12a-PCV3 detection targeting PCV3 Cap DNA (2 × 10 9 copies/µL).The results showed that the UV light, blue light, and fluorescence signal increased with time and peaked at 40 min in the CRISPR/Cas12a-PCV3 detection stage, whereas the control group did not show any positive signal (Figure 1A,B).A positive fluorescence signal was observed after 5 min; therefore, we artificially set the detection time to 10 min based on the timeliness of this detection method.Finally, the total time for this two-pot detection method was set to 30 min (RPA for 20 min and the CRISPR reaction not exceeding 10 min) (Figure 1C).not show any positive signal (Figure 1A,B).A positive fluorescence signal was observed after 5 min; therefore, we artificially set the detection time to 10 min based on the timeliness of this detection method.Finally, the total time for this two-pot detection method was set to 30 min (RPA for 20 min and the CRISPR reaction not exceeding 10 min) (Figure 1C).
The specificity of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method was further analyzed.Viral DNA or RNA of nine swine viruses were confirmed by conventional PCR or RT-PCR (Figure 3A).Porcine circoviruses have four genotypes, namely PCV1, PCV2, PCV3, and PCV4 [2], prompting us to determine the specificity of this method for various genotypes.The extracted genomes from the four PCV genotypes were detected, and the results showed that only the PCV3 genome exhibited a positive UV light, blue light, and fluorescence signal, whereas the other viral genotypes did not (Figure 3B,C).Subsequently, the specificity of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method for DNA or RNA viruses (PRV, PRRSV, SVA, PEDV, and CSFV) was analyzed.As shown in Figure 3D,E, the PCV3-positive sample, instead of the other samples, showed a specific UV light, blue light, and fluorescence signal.Together, these results indicate that the two-pot RPA-CRISPR/Cas12a-PCV3 detection method is highly sensitive and specific for PCV3.The specificity of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method was further analyzed.Viral DNA or RNA of nine swine viruses were confirmed by conventional PCR or RT-PCR (Figure 3A).Porcine circoviruses have four genotypes, namely PCV1, PCV2, PCV3, and PCV4 [2], prompting us to determine the specificity of this method for various genotypes.The extracted genomes from the four PCV genotypes were detected, and the results showed that only the PCV3 genome exhibited a positive UV light, blue light, and fluorescence signal, whereas the other viral genotypes did not (Figure 3B,C).Subsequently, the specificity of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method for DNA or RNA viruses (PRV, PRRSV, SVA, PEDV, and CSFV) was analyzed.As shown in Figure 3D,E, the PCV3-positive sample, instead of the other samples, showed a specific UV light, blue light, and fluorescence signal.Together, these results indicate that the two-pot RPA-CRISPR/Cas12a-PCV3 detection method is highly sensitive and specific for PCV3.

Evaluating Consistency Between Two-Pot RPA-CRISPR/Cas12a-PCV3 Detection Method and Real-Time Quantitative PCR (qPCR) Method
To evaluate the performance of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method, 25 clinical samples, including spleen, lung, kidney, submandibular lymph node, and inguinal lymph node, were analyzed.As shown in Table 1 and Figure 4A, 18 of 25 clinical samples were positive, while the remaining samples were negative.To verify the reliability of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method for the detection of PCV3, a previously published qPCR method was used to verify the above detection results.The detection results of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method and qPCR were consistent (Figure 4B), with a 100% coincidence rate, suggesting that the two-pot RPA-CRISPR/Cas12a-PCV3 detection method is reliable.

Evaluating Consistency between Two-Pot RPA-CRISPR/Cas12a-PCV3 Detection Method and Real-Time Quantitative PCR (qPCR) Method
To evaluate the performance of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method, 25 clinical samples, including spleen, lung, kidney, submandibular lymph node, and inguinal lymph node, were analyzed.As shown in Table 1 and Figure 4A, 18 of 25 clinical samples were positive, while the remaining samples were negative.To verify the reliability of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method for the detection of PCV3, a previously published qPCR method was used to verify the above detection results.The detection results of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method and qPCR were consistent (Figure 4B), with a 100% coincidence rate, suggesting that the two-pot RPA-CRISPR/Cas12a-PCV3 detection method is reliable.

The Clinical Samples Detection of One-Pot RPA-CRISPR/Cas12a-PCV3 Method
To simplify the manipulation procedure of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method and reduce the risk of secondary operations or aerosol contamination [22], the two-pot detection method was further optimized.We combined the RPA amplification step and the CRISPR/Cas12a-PCV3 detection step into one tube [23], in which the reagents for

The Clinical Samples Detection of One-Pot RPA-CRISPR/Cas12a-PCV3 Method
To simplify the manipulation procedure of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method and reduce the risk of secondary operations or aerosol contamination [22], the two-pot detection method was further optimized.We combined the RPA amplification step and the CRISPR/Cas12a-PCV3 detection step into one tube [23], in which the reagents for RPA amplification and CRISPR/Cas12a-PCV3 detection were added to the bottom and cap of the tube, respectively (Figure 5A).After RPA amplification, the reagents were rotated to initiate CRISPR/Cas12a detection.The results showed that the minimum detection limit of this one-pot detection method could reach 2 × 10 1 copies/µL (Figure 5B,C).To determine a more accurate detection limit, the refined viral DNA (concentrations 2 × 10 1 , 1 × 10 1 , 0.5 × 10 1 , and 0.25 × 10 0 copies/µL) were further detected.The results showed that the minimum detection limit of the one-pot detection method was 0.5 × 10 1 copies/µL (5 copies/µL) (Figure 5D,E).Subsequently, 25 clinical samples were analyzed using the one-pot method to evaluate their performance.The results showed that 18 out of 25 clinical samples were positive, and the remaining samples were negative (Figure 6A,B), suggesting that the one-pot RPA-CRISPR/Cas12a-PCV3 detection method is feasible.
Animals 2024, 14, x FOR PEER REVIEW 10 of 14 RPA amplification and CRISPR/Cas12a-PCV3 detection were added to the bottom and cap of the tube, respectively (Figure 5A).After RPA amplification, the reagents were rotated to initiate CRISPR/Cas12a detection.The results showed that the minimum detection limit of this one-pot detection method could reach 2 × 10 1 copies/µL (Figure 5B,C).To determine a more accurate detection limit, the refined viral DNA (concentrations 2 × 10 1 , 1 × 10 1 , 0.5 × 10 1 , and 0.25 × 10 0 copies/µL) were further detected.The results showed that the minimum detection limit of the one-pot detection method was 0.5 × 10 1 copies/µL (5 copies/µL) (Figure 5D,E).Subsequently, 25 clinical samples were analyzed using the one-pot method to evaluate their performance.The results showed that 18 out of 25 clinical samples were positive, and the remaining samples were negative (Figure 6A,B), suggesting that the one-pot RPA-CRISPR/Cas12a-PCV3 detection method is feasible.

Discussion
PCV3 infections have been reported worldwide, causing huge economic losses to the swine industry.The early diagnosis of various pathogens plays an important role in the prevention and control of related diseases.Nucleic acids, which are the most conventional detection targets, have been used for the diagnosis and detection of various pathogens, which has prompted nucleic acid amplification, such as PCR-related techniques, to become a sensitive and specific method [10,11].However, these techniques have limited application owing to expensive instrumentation and complex operation procedures.Thus, a better method that overcomes these shortcomings is urgently required for pathogen detection.In this study, we developed a nucleic acid detection method targeting

Discussion
PCV3 infections have been reported worldwide, causing huge economic losses to the swine industry.The early diagnosis of various pathogens plays an important role in the prevention and control of related diseases.Nucleic acids, which are the most conventional detection targets, have been used for the diagnosis and detection of various pathogens, which has prompted nucleic acid amplification, such as PCR-related techniques, to become a sensitive and specific method [10,11].However, these techniques have limited application owing to expensive instrumentation and complex operation procedures.Thus, a better method that overcomes these shortcomings is urgently required for pathogen detection.In this study, we developed a nucleic acid detection method targeting the PCV3 Cap gene, the RPA-CRISPR/Cas12a-PCV3 detection method, by combining the RPA and CRISPR/Cas12a techniques, in which crRNA activates Cas12a endonuclease activity by recognizing specific target sequences, followed by cleaving an ssDNA-FQ probe to produce a fluorescent signal for PCV3 diagnosis.
To date, some isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP) [24], enzymatic recombinase amplification (ERA) [25], and RPA [26], have been developed as improved methods over conventional PCR methods, based on their simplicity, rapidity, and low cost.However, the emergence of nonspecific amplification signals in these detection methods limits their application [27].Recently, the combination of crRNA-mediated CRISPR/Cas endonuclease targeting nucleic acid detection techniques has enhanced the specificity of isothermal amplification methods [28,29].This high specificity is attributed to the specific recognition of PAM sites by the Cas protein in the presence of crRNA [30].Cas12a performs nonspecific cleavage of ssDNA by recognizing specific dsDNA sequences, which is suitable for the detection of dsDNA viruses such as PCV3 and ASFV.
The CRISPR/Cas12a detection conditions, such as Cas12a endonuclease activity, probe concentration, Cas12a/crRNA ratio, and detection sensitivity of the Cas12 enzyme, were optimized according to the results of the fluorescence signal (Figures S1 and S3).The Cas12a enzyme possesses only low detection sensitivity without pre-amplification because of its weak cleavage activity [12,31].Our results confirmed the low sensitivity of Cas12a and showed that the detection sensitivity of the Cas12a enzyme itself was only 2 × 10 9 copies/µL viral DNA (Figure S3B,C), which verified the character of the low sensitivity of Cas12a activity.Thus, CRISPR/Cas12a detection must be combined with isothermal amplification to enhance sensitivity.We established a two-pot RPA-CRISPR/Cas12a-PCV3 detection method (Figure 1), which not only shortens the detection time but also improves detection sensitivity, with a minimum detection limit of two copies/µL of viral DNA (Figure 2).The detection sensitivity of this method is superior to that of previously reported methods, such as the TagMan-based real-time PCR [32], LAMP [33], and RPA assays [34].The specificity of this method did not show any cross-positive reactions targeting other viruses (Figure 3B-E), which is important for clinical detection.Subsequently, the coincidence rate of the 25 clinical samples detected between the two-pot RPA-CRISPR/Cas12a-PCV3 detection method and the previously reported qPCR method was 100% (Figure 4), and all positive samples were further validated by sequencing, suggesting the reliability and utility of the two-pot method.We further integrated the amplification and detection steps into a single tube to simplify the operation and reduce pollution (Figure 5).Our established one-pot detection method reached a sensitivity limit of five copies/µL viral DNA, and its detection results of the clinical samples were completely consistent with the two-pot method (Figure 6), indicating that the one-pot method is reliable and suitable for a clinical test.

Conclusions
In summary, we successfully developed one-and two-pot RPA-CRISPR/Cas12a-PCV3 detection methods that could detect PCV3 within 30 min at a minimum detection limit of five and two copies of viral DNA, respectively.These new methods with high sensitivity, specificity, and short assay times provide advantages for virus detection and are suitable for the prevention of the occurrence and spread of PCV3 at an early stage.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani14172527/s1, Figure S1.The expression and purification of Cas12a proteins.Figure S2.The validation of three crRNAs and optimization of the Cas12a/crRNA concentration in CRISPR/Cas12a reaction.Figure S3.The validation of RPA primers and the sensitivity determination of the CRISPR/Cas12a reaction.Table S1.Primers, crRNA, and probe in this study.

Figure 4 .
Figure 4. Detection of PCV3 clinical samples by two-pot RPA-CRISPR/Cas12a-PCV3 detection method.(A) The 25 clinical samples detection of two-pot RPA-CRISPR/Cas12a-PCV3 detection method by blue light, and UV light.(B) Comparison of detection results using two-pot RPA-CRISPR/Cas12a and qPCR-based PCV3 detection methods, with blue indicating negative results and orange indicating positive results.NTC represents no-viral DNA control.

Figure 4 .
Figure 4. Detection of PCV3 clinical samples by two-pot RPA-CRISPR/Cas12a-PCV3 detection method.(A) The 25 clinical samples detection of two-pot RPA-CRISPR/Cas12a-PCV3 detection method by blue light, and UV light.(B) Comparison of detection results using two-pot RPA-CRISPR/Cas12a and qPCR-based PCV3 detection methods, with blue indicating negative results and orange indicating positive results.NTC represents no-viral DNA control.

Figure 6 .
Figure 6.Detection of PCV3 clinical samples by one-pot RPA-CRISPR/Cas12a-PCV3 detection method.(A) The 25 clinical samples detection of one-pot RPA-CRISPR/Cas12a-PCV3 detection method by blue light and UV light.(B) Comparison of detection results between one-and two-pot RPA-CRISPR/Cas12a detection methods, with blue indicating negative results and orange indicating positive results.NTC represents no-viral DNA control.

Figure 6 .
Figure 6.Detection of PCV3 clinical samples by one-pot RPA-CRISPR/Cas12a-PCV3 detection method.(A) The 25 clinical samples detection of one-pot RPA-CRISPR/Cas12a-PCV3 detection method by blue light and UV light.(B) Comparison of detection results between one-and two-pot RPA-CRISPR/Cas12a detection methods, with blue indicating negative results and orange indicating positive results.NTC represents no-viral DNA control.