A total of 33 P. sojae isolates from different geographic areas were tested in this study, as well as 37 isolates from 17 different oomycetes (including 34 isolates of 14 species representing 10 clades of Phytophthora), and 16 other fungi and bacteria isolated from soybean and other hosts. The origin, host affiliation, and number of tested isolates are listed in Table 1. All isolates were routinely maintained in liquid nitrogen at the School of Breeding and Multiplication, Hainan University, China.

Genomic DNA was extracted from mycelial cultures using a DNAsecure Plant kit (Tiangen, Beijing, China) as described previously (Wu et al., 2017). The purified genomic DNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific), and stored as aliquots at a concentration of 100 ng μL⁻¹ in sterile distilled water at −20°C.

All the primers, DNA fragments (including CRISPR RNA [crRNA], and ssDNA probe) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). REnGen® Lba Cas12a and NEBufferTM were purchased from New England BioLabs Ltd. (Beijing, China). High-Affinity HotStart Taq (ET108-1) and 2 × PCR SuperMix were purchased from Tiangen Biotech (Beijing, China), while RPA Kits were obtained from AMP-Future Biothech Co., Ltd. (Changzhou, China). The lateral flow strips were purchased from Tiosbio Biotech Co. Ltd. (Beijing China). Nuclease-free H₂O was obtained from Sangon Biotech (Shanghai, China). Genomic DNA Kits were purchased from Tiangen Biotech (Beijing, China).

To identify the conserved, single-copy, candidate detection targets for P. sojae specific PCR or RPA assays, we retrieved the annotated genomic sequence of P. sojae.¹ All the gene sequences of P. sojae were used to perform BLAST searches against the publicly available genomic sequences of Phytophthora and related taxa (e.g., Pythium, and Phytopythium species) in the National Center for Biotechnology Information (NCBI) database. A total of 1,187 P. sojae genes did not share any sequence similarity with genomic sequences of any other species. Three loci (PHYSODRAFT_299276, PHYSODRAFT_255386, and PHYSODRAFT_531894) were randomly selected as detection targets to test the specificity using a collection of Phytophthora species (Table 1) in conventional PCR. Ultimately, a single-copy gene (PHYSODRAFT_299276) was selected as the specific target for P. sojae detection.

The target sequence of P. sojae (PHYSODRAFT_299276) was used to design the crRNAs and primers for the CRISPR/Cas assay. RPA primers were designed and analyzed using Primer 5 and Primer-Explore V5 software.² The crRNA with 20–24 bp target-dependent sequence following PAM (5’-TTTN-3′) sit was designed using EuPaGDT ³ and an RNA scaffold to assist in protein binding. RPA primers and crRNA were synthesized by Sangon Biotech (Shanghai, China). The sequences of all primers are listed in Supplementary Table S1.

Conventional PCR assays were performed in 10 μL reactions. Each reaction included 5 μL of 2 × Taq PCR Master Mix (Tiangen Biotech Co., Ltd., Beijing, China), 0.5 μL of each forward and reverse primer (10 μM), 1 μL of template DNA (100 ng μL⁻¹), and 3.0 μL of nuclease-free H₂O. Thermal cycling was carried out using PTC-200 Thermo Cycler (MJ Research, Watertown, MA, United States) with the following conditions: 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 58°C for 45 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR amplification products were separated by electrophoresis on 2.0% agarose gel, stained with ethidium bromide, and visualized under UV light. Each experiment was performed in triplicate.

RPA assay was performed using the AMP-Future Biotech Co. Ltd. (Changzhou, China) according to the manufacturer’s instructions. Briefly, each 10 μL reaction contained 5.9 μL of A buffer, 0.5 μL of B buffer, 0.4 μL of each forward and reverse primers (10 μM), 1 μL of DNA (100 ng μL⁻¹), and 0.8 μL of nuclease-free H₂O. The mixture was incubated in a conventional water bath at 38°C for 15 min for RPA reaction.

The efficiency of the CRISPR/Cas12a cleavage system is directly influenced by the concentration of crRNA and Cas12a and affects both trans-cleavage efficiency and fluorescence intensity. To determine the optimal CRISPR/Cas12a reaction conditions, a range of Cas12a and crRNA concentrations (50 nM ~ 300 nM), Cas12a-mediated cleavage temperature (37 ~ 42°C), RPA reaction time (5 min ~ 30 min), and trehalose concentrations (0.006 mM ~ 6 mM) were tested. Nuclease-free water was used as a negative control. The CRISPR/Cas12a reaction products were analyzed by naked eye observation or by detecting the maximal fluorescence signal value to determine the optimal CRISPR/Cas reaction conditions. All experiments were repeated three times.

The CRISPR/Cas12a reaction was performed in a 5 μL reaction mixture containing 0.75 μL crRNA (4 μM), 1.5 μL Cas12a (2 μM), 0.75 μL ssDNA FQ reporter (100 nM), and 1.5 μL of 10× NEB Buffer r2.1, 0.5 μL of nuclease-free H₂O, along with 10 μL of RPA products (or 0.5 μL of 6 mM trehalose and 10 μL of PCR products). In the PCR-CRISPR/Cas12a assay, 6 mM trehalose was added to the reaction solution to protect the activity of the Cas12a protein. The reactions were incubated at 38°C for 10 min and then placed on an ultraviolet (UV) or blue light transilluminator for naked-eye detection.

The one-pot CRISPR/Cas12a detection combines RPA/PCR reaction with Cas12a cleavage in a single tube reaction. To perform the assay, 10 μL of the RPA/PCR assay containing targets was added to the bottom of the tube, and 5 μL of CRISPR/Cas12a reaction solution was initially added inside the inner wall of the tube lid. After incubating the RPA/PCR reaction for 10–60 min, the CRISPR/Cas12a solution was swung into the RPA reaction solution by hand on-site, and the mixture was incubated for an additional 15 min. The reaction tubes were then placed on a blue or UV light transilluminator for naked eye detection (Figure 1).

The structure of the lateral flow strip (LFS) incorporated an absorbent pad, a conjugate pad, a nitrocellulose (NC) membrane, and a sample pad on a plastic adhesive backing card. The capture reagents streptavidin and anti-IgG antibody were dispensed onto the NC membrane, and each band was separated by 5 mm. When there is no target in the system, the conjugate was captured by the streptavidin through biotin, the control line became red because of the aggregation of the colloidal gold in this area, while the test line without the flow-through of the conjugate had no color change. However, if there are targets in the system, the ssDNA reporter is cleavaged by CRISPR/Cas12a, and FAM and biotin molecules are separated. After the integration of ssDNA-biotin captured by streptavidin at the C line, the remaining conjugate of FAM-ssDNA and anti-FAM antibody would flow into the test line and be captured by anti-lgG antibody. The lateral flow detection was performed according to the Tiosbio protocol (Tiosbio Biotech Co. Ltd., Beijing China). Briefly, 85 μL Tiosbio assay buffer was added to 15 μL of the RPA-CRISPR/Cas12a reaction and incubated for 5 min. The lateral flow test strip was then inserted into the reaction mixture for 5 ~ 10 min to allow the reaction to occur. A positive test result was indicated by the appearance of both the control and test lines, while a negative result showed only the control line. All lateral flow assays were performed in triplicate.

To test the specificity of the one-pot RPA/PCR CRISPR/Cas12a assays, a total of 33 P. sojae isolates from different geographic origins, 37 isolates from 17 different oomycetes species, and 16 other fungi and bacteria were used (Table 1). To determine the sensitivity of the CRISPR/Cas12a assay, 10-fold serial dilutions of P. sojae purified gDNA ranging from 100 to 0.001 ng μL⁻¹ were used as DNA templates. Nuclease-free H₂O was used as the negative control. All assays were repeated in triplicate for each concentration of the gDNA template.

To assess the practical application of the one-pot CRISPR/Cas12a detection for P. sojae, soybean seedlings were inoculated with P. sojae as described previously (Zhao et al., 2015). The DNA was extracted and evaluated using the developed RPA/PCR-CRISPR/Cas12a assays. Each experiment was repeated three times. In addition, a total of 20 soybean plants collected from different fields in Fujian and Hainan provinces were assayed using the RPA/PCR-CRISPR/Cas12a assays. The DNA was extracted from infected soybeans as described previously (Chen et al., 2013), and the RPA/PCR-CRISPR/Cas12a assay was performed as described above. Purified DNA of P. sojae was used as the positive control and healthy soybeans were used as negative control. The P. sojae infection was confirmed using the conventional isolation culture method for the identification of the pathogens based on morphological characteristics. Each experiment was repeated three times.

A schematic description of the Cas-OPRAD assay and the approximate time of each step are shown in Figure 1. The shortest time of Cas-OPRAD assay can reach about 0.5–1 h, including three primary steps: sample preparation, Cas-OPRAD assay, and visual readout. In the first step, the sample was prepared by using a rapid nucleic acids extraction method, and then the RPA or PCR technique ensured the efficient production of DNA amplification from the sample. In the first step, the sample was prepared by using a rapid nucleic acids extraction kit, the RPA or PCR technique was employed to enrich the target DNA fragments, which ensures the efficient production of DNA amplification from the sample. We utilized the principle of liquid surface tension and spatial isolation to separate the RPA/PCR reaction from sgRNA-Cas12 in a one-pot reaction. Specifically, the sgRNA-Cas12 complex was adsorption suspended on the top cover of the reaction tube, while the RPA or PCR reaction was located at the bottom of the tube. After the nucleic amplification reaction was completed, a simple centrifugation or manual shaking was used to mix the two components for reaction, this design avoids cross-contamination of various components in complex systems. For amplification product identification, the gRNA was specially designed to target RPA/PCR products, and the amplification products were recognized and cleaved, especially by the sgRNA and Cas12a complex. Furthermore, the Cas12a-sgRNA-target ternary complex activated the trans-cleavage activity of Cas12a, resulting in ultrafast cleavage of the ssDNA fluorescent probe and consequent fluorescent signal generation. The fluorescent signal generated from trans-cleavage was monitored by a fluorescence detector or LFS device.

The specificity of Cas OPRAD was carried out in three steps. Firstly, we determine sequence specificity through comparative genomic analysis, 1,187 candidate genes were identified from P. sojae. Pair-wise gene sequence alignments were then performed, and three P. sojaespecific genes were selected as the detection target candidates: PHYSODRAFT_299276, PHYSODRAFT_255386, and PHYSODRAFT_531894. Then, the primers were designed for these genes, and a conventional PCR assay was used to evaluate the specificity of the primer pairs. Primers So-F/So-R (Supplementary Table S1), designed from PHYSODRAFT_299276, successfully distinguished P. sojae from other plant pathogens by amplifying a 1,310 bp target band. In contrast, PHYSODRAFT_255386 and PHYSODRAFT_531894 amplified non-target bands from the gDNA of P. sojae (data not shown). These results suggest that the primers designed from PHYSODRAFT_299276 had good specificity for P. sojae detection. Therefore, a probe for PHYSODRAFT_299276 was used as the detection target.

To verify the feasibility of our scheme, we first optimized the concentration of crRNA and the ratio of Cas12a to crRNA in the CRISPR/Cas12a assay, as the crRNA concentration plays a crucial role in CRISPR/Cas12a reaction. The optimal concentration of crRNA and the optimal ratio of Cas12a to crRNAs were determined to be 200 nM and 1:1, respectively (Supplementary Figure S1A). Under the optimized conditions of crRNA concentration and the ratio of Cas12a to crRNAs, we further optimized CRISPR/Cas12a reaction time and temperature. The ideal conditions for the cleavage were determined to be 38°C for 10 min (Supplementary Figures S1B,C).

To maintain the activity of Cas12a in one pot, according previous report by Saisawang et al. (2023), we adjusted the temperature of the heat cap in the PCR-CRISPR/Cas12a reaction to 50°C and trehalose was added into CRISPR/Cas reaction to help enzyme stabilization in target cleavage. The optimized concentration of trehalose for PCR-CRISPR/Cas12a was 0.6 mM (Supplementary Figure S1D). Therefore, subsequent PCR-CRISPR/Cas12a assays were carried out under these conditions. We tested eight reaction systems (reactions # 1–8) for PCR-CRISPR/Cas12a (Supplementary Figure S2) and six reactions (reactions # 1–6) for RPA-CRISPR/Cas12a, respectively (Figure 2). The generation of fluorescence comes from Cas12 cleavage FQ ssDNA probe molecules, after incubation at 38°C for 15 min and 5 ~ 10 min of CRISPR/Cas detection, only reaction #1, containing Cas12a, crRNAs, target DNA, ssDNA-FQ, and RPA or PCR reaction mixture, generated a naked-eye visual result that was visible under blue or UV light (Figure 2; Supplementary Figure S2). To further confirm the specificity and reaction time of the fluorescence signal, the one-pot RPA-CRISPR/Cas12a assay was analyzed on an AriaMx quantitative PCR instrument. These fluorescence value results demonstrate that the CRISPR/Cas12a assay provides a high fluorescence intensity value (>30,000 AU) within 15 min, which indicates that Cas-OPRAD can be offered a rapid, specific, and simple method for detecting P. sojae (Figure 2B).

To validate the specificity of Cas OPRAD detection, DNA templates were extracted from 33 P. sojae isolates and 53 isolates of other non-P. sojae were evaluated (Table 1). After the Cas-OPRAD reaction, only P. sojae showed a positive reaction, while no color change was observed for the other pathogens, including other Phytophthora spp. and other fungal and bacteria isolates from soybeans (Figures 3A,B; Supplementary Figures S3A,B). Notably, there was no cross-reaction with closely related species, such as P. melonis, P. cinnamomi, and P. vignae, which belong to the same clad 7b as P. sojae. These results demonstrate that the Cas-OPRAD assay developed in this study is highly specific for P. sojae.

To further evaluate the sensitivity of the CRISPR/Cas12a assay, 10-fold serially diluted DNA templates of P. sojae were used. As shown in Supplementary Figures S4A,B the detection limit for the PCR-CRISPR/Cas12a was 100 pg. μL⁻¹, while the sensitivity of the RPA-CRISPR/Cas12a assay reached 10 pg. μL⁻¹ in both fluorescence detection (Figures 3C,D). Notably, the sensitivity for Cas-OPRAD was found to be at least 10 times higher than that of the PCR-CRISPR/Cas12a method. These results suggest that the RPA-CRISPR/Cas12a-based assay is more sensitive for early detection of the soybean pathogen P. sojae.

The Cas-OPRAD assay is performed at an isothermal temperature, making it potentially suitable for on-site detection. In addition to fluorescence detection, we also utilized lateral flow strips (LFS) for a visual CRISPR/Cas12a assay that can facilitate on-site detection of P. sojae. The principle of the test strip is shown in Figure 4A, the structure of LFS incorporated an absorbent pad, a conjugate pad, a nitrocellulose (NC) membrane, and a sample pad on a plastic adhesive backing card. The capture reagents streptavidin and anti-IgG antibody were dispensed onto the NC membrane, and each band was separated by 5 mm. When there is no target in the system, the conjugate was captured by the streptavidin through biotin, the control line became red because of the aggregation of the colloidal gold in this area, while the test line without the flow-through of the conjugate had no color change. However, if there are targets in the system, the ssDNA reporter is cleavaged by CRISPR/Cas12a, and FAM and biotin molecules are separated. After the integration of ssDNA-biotin captured by streptavidin at the C line, the remaining conjugate of FAM-ssDNA and anti-FAM antibody would flow into the test line and be captured by anti-lgG antibody. As expected, bands appeared on both the test line and control line of the strip in a positive reaction, while only a band appeared on the control line in a negative reaction. Therefore, the lateral flow assay format provides a rapid, simple, instrument-free, on-site field detection method for P. sojae.

The specificity and sensitivity of the Cas-OPRAD LFS platform were also evaluated for the detection of P. sojae. The specificity test results were visualized using LFS tests (Figure 4B). In the LF-OPRAD assay, red bands at the T line were only observed for the Cas-OPRAD products of the P. sojae DNA sample, and no red band at the T line was observed for reactions with other pathogens. These results indicate that the developed Cas-OPRAD platform for the P. sojae pathogens was highly specific. In sensitivity tests, the results from LFS indicated are same sensitivity as for the Cas-OPRAD assays. For example, a red band at the T line was observed for reactions at all P. sojae DNA dilutions except for 1 pg. μL⁻¹ and NC (Figure 4C), indicating that the sensitivity of Cas-OPRAD was 10 pg. μL⁻¹, these results indicate that the on-site platform could be used for P. sojae pathogen detection without dedicated professional equipment.

To verify the validity and feasibility of the Cas-OPRAD for diagnosing soybean samples, the DNA was extracted from three regions (I: roots and underground stems. II: cotyledons and stem above the part of ground. III: epicotyl and first leaf) of inoculated soybean seedlings (Figure 5A). As shown in Figures 5B–D, positive reactions were observed in regions I and II, but not in region III or healthy soybeans using Cas-OPARD or PCR-CRISPR/Cas12a, the result indicates that under artificial inoculation, the P. sojae infection in the roots of soybeans is higher than that in the leaves. Therefore, in field sample collection, it is necessary to prioritize collecting regions I or II for Cas-OPARD analysis.

According to the results above, a total of 20 field soybean samples collected from natural fields of Fujian and Haina province, China, were used for Cas-OPARD assay or PCR-CRISPR/Cas12a with both fluorescence and lateral flow readouts. Of the 20 collected samples, 16 were positive and four were negative using both the Cas-OPARD, the results obtained by using the PCR-CRISPR/Cas12a method are consistent with those obtained by Cas-OPRAD (Figures 5E,F). The detection results showed 100% consistency between the visual detection under blue or UV light and the lateral flow strip readout (Figures 5F,G). These results demonstrate the high detection capabilities of the CRISPR/Cas12a assay for P. sojae.