Recombinase Polymerase Amplification/Cas12a-Based Identification of Xanthomonas arboricola pv. pruni on Peach

Peach bacterial spot caused by Xanthomonas arboricola pv. pruni (Xap) is a devastating disease worldwide and frequently causes massive economic losses. In recent years, it has become a pandemic outbreak in most peach production areas of China, especially on precocious peaches in the middle reach of the Yangtze River. Rapid, user-friendly detection is extremely important to make the correct diagnosis and develop suitable control strategies. In this study, we described a recombinase polymerase amplification (RPA)/Cas12a-based system that combines RPA and CRISPR/Cas12a for Xap identification. A total of three crRNAs were designed to target a highly conserved ABC transporter ATP-binding protein-encoding gene ftsX to make specific detection of Xap. Results showed that crRNA 2 and crRNA 3 could get consistent detection for Xap. To realize the visualization of detection results, we additionally introduced FQ-reporter and FB-reporter. The developed method was highly sensitive and could detect as low as 10^–18 M Xap gDNA with a mini-UV torch, corresponding to 1.63 copies/μl or 8.855 fg/μl gDNA of Xap, while with lateral flow strips, the sensitivity was 10^–17 M. In addition, this method could specifically detect Xap from other closely related bacteria or pathogens associated with peach diseases. Furthermore, this method could make correct identification for Xap with crude DNA using NaOH-based extraction (3 min) directly from diseased peach samples. Considering that the developed method could get results within 2 h and could be performed at 37°C (body temperature), it is promising to be applied for Xap diagnosis and monitoring in fields.

Introduction

Bacterial spot caused by Xanthomonas arboricola pv. pruni (Xap) is an economically important disease of peach worldwide and results in huge economic losses on most of the stone fruit trees (Stefani, 2010; Janse, 2012). In recent years, it has become a widespread epidemic issue that broke out into disasters in some orchards, especially on precocious peaches in the middle reach of the Yangtze River where the disease incidence can reach 100% on susceptible cultivars. Xap infects not only leaves but also fruits and branches on a wide range of Prunus species including peach (Prunus persica), plum (Prunus salicina), apricot (Prunus armeniaca), cherry (Prunus avium), and almond (Prunus amygdalus), as well as ornamental plants such as Prunus davidiana and Prunus laurocerasus (Rosello et al., 2012; Tjou-Tam-Sin et al., 2014). The initial symptom on leaves is the appearance of water-soaked spots, then the spots gradually enlarge and become necrotic in the center, eventually falling off to form irregular perforations, and even leading to leaf fall. On fruits, diseased tissue presents deep and wide-shaped cracking, which seriously affects the quality and taste of peaches. These characteristics share similar clinical signs with the peach scab, black spot, and early canker (Bock et al., 2011; Oura et al., 2013), making its diagnosis difficult. Besides, a low isolation rate and similar colony characteristics to other epiphytic bacteria are also obstacles to its diagnosis. Consequently, diagnostic errors occur frequently.

The current recommended international standard for Xap diagnosis according to the European and Mediterranean Plant Protection Organization (EPPO) is based on pathogen isolation (European and Mediterranean Plant Protection Organization [EPPO], 2006), which is accurate, but laborious and time-consuming. With the development of molecular technology, new molecular detection tools have been invented for nucleic acid detection, such as PCR, bio-PCR (Park et al., 2010), duplex PCR (Pothier et al., 2011a), multiplex PCR (Pothier et al., 2011b), and real-time PCR (Palacio-Bielsa et al., 2011). Nevertheless, such methods have limitations in realizing visual-readout way, thus are not suitable for local diagnosis (Bai et al., 2019). Recently, isothermal amplification technology started to be popular and made a new focus in nucleic acid detection due to its simplicity and accuracy, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). However, LAMP requires a relatively higher reaction temperature (around 60°C) and is prone to high false positives, which affects its field use (Suleman et al., 2016). RPA is the latest developed isothermal amplification detection technology in the world and is claimed to eventually replace PCR. RPA targets the double-stranded DNA (dsDNA) by recombinase-primer complex and amplifies the target region through strand-displacement DNA synthesis (Piepenburg et al., 2006). It can successfully amplify targeted DNA sequences at 37–42°C for 30 min with high sensitivity. The results can be visualized by combining them with fluorescence signals or lateral flow assay (LFA).

Recently, another new nucleic acid detection tool emerged and switched to a new wave of scientific research. It is the RNA-guided CRISPR/Cas-based nucleic acid detection system that has shown great promise for highly sensitive and rapid nucleic acid detection (Sashital, 2018; Li et al., 2019a). In this system, three nucleases, namely, Cas12a, Cas13a, and Cas14, were generally used (Gootenberg et al., 2017, 2018; Chen et al., 2018; Harrington et al., 2018; Li et al., 2018; Teng et al., 2019). The specially designed crRNA complementary to the target can activate the cis- and trans-cleavage activities of Cas nuclease, which not only cleave dsDNA target in a specific way but also cleave the non-specific single-stranded DNA (ssDNA) in the whole environment. The CRISPR/Cas system is also evolving into many innovative applications including nucleic acid detection, such as DETECTR, SHERLOCK, and HOLMES (Gootenberg et al., 2017, 2018; Knott and Doudna, 2018; Myhrvold et al., 2018; Li et al., 2019a). They are generally combined with RPA, LAMP, or PCR to amplify the targeting signal. The Cas12a-based DETECTR and HOLMES target dsDNA and detect the signal with fluorescence detection equipment, thus limiting the application under field conditions (Li et al., 2018). Cas13a-based SHERLOCK, both the target and probe are RNAs, which requires an additional in vitro transcription step to convert dsDNA to RNA (Gootenberg et al., 2017, 2018). Cas14 targets ssDNA, which also needs extra conversion from dsDNA to ssDNA (Harrington et al., 2018). Overall, Cas12a-based detection has more potential to be used under field conditions with some modifications. Actually, Bai et al. (2019) developed the CORDS (Cas12a-based on-site and rapid detection system), which combined Cas12a with recombinase aided amplification (RAA) and an immunochromatographic lateral flow strip for ASFV on-site detection. Zhang et al. (2020) also used Cas12a combined with RPA and lateral flow strip to detect rice blast pathogen.

In this study, we applied the Cas12a-based system combined with RPA and an ssDNA probe to detect Xap, the causal agent of peach bacterial spot. Two visualization approaches were tailored, one is the fluorescence visualization readout and the other is to use lateral flow strips. We found a mini-UV torch, which can detect the fluorescence signal easily instead of the complicated fluorescence detection instrument. The integrated RPA/Cas12a detection procedure was conducted for Xap detection from diseased and healthy samples. It could make an accurate detection even using the crude DNA with NaOH-based DNA quick extraction, and the whole detection only took approximately 1 h. As an easy, effective, and field-deployable application, it is anticipatory that this innovative nucleic acid detection method using RPA/Cas12a will be a useful tool for early detection of Xap in practice.

Materials and Methods

Isolates and Plant Materials

Xanthomonas arboricola pv. pruni isolate ZY2-3-1a was routinely used in Xap detection assays. Two closely related Xanthomonas species/subspecies, namely, Xanthomonas campestris pv. campestris and X. arboricola pv. juglandis, other nine bacterial species which were isolated and identified from peach samples, and four common fungal species, namely, Alternaria alternata, Venturia carpophila, Monilinia fructicola, and Wilsonomyces carpophilus from peach were used in the assessment of the specificity of RPA/Cas12a assay (Supplementary Table 1). All the isolates were identified to the species level based on their morphological characteristics and molecular identification (comparison of ITS sequences for fungal species and 16S rDNA for bacterial species). For RPA/Cas12a assay in field detection, the peach leaf samples were collected from the cultivar Xiahui 5 at an orchard in Huazhong Agricultural University, Wuhan, Hubei Province, China.

DNA Preparation

For regular genomic DNA preparation, the Rapid Bacterial Genomic DNA Isolation Kit (ToloBio, Shanghai, China) was used to purify DNA according to the instructions of the manufacturer.

For field rapid detection, the NaOH-based crude DNA extraction was used, and 2–4 pieces of diseased or healthy tissues (around 0.6 cm × 2 cm, 50 mg) were ground in a mortar and submerged in 500 μl of 0.5 M NaOH (Qian et al., 2018). After it was blended for 1 min and kept at room temperature for 1–2 min, the supernatant was diluted 50-fold with Tris-EDTA (TE) buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and directly used as a template for the RPA/Cas12a assay.

Cas12a crRNA Design and Synthesis

CrRNA guide sequence was designed according to the adjacent motif of PAM (TTTV) for the ftsX gene which has been described as a Xap specific gene in the previous study (Pothier et al., 2011a). In brief, the crRNAs were designed based on the targeting sequence (a 323 bp fragment of ftsX gene) at the website https://portals.broadinstitute.org/gppx/crispick/public by simply selecting corresponding PAM sites and Cas12a and then, select candidates based on the resulted scores. For the synthesis of the crRNAs, a DNA oligo (100 μM) (T7-crRNA-oligo) containing T7 promoter, guide sequence, and conserved stem-loop sequence was synthesized at Tianyi Huiyuan, Wuhan Biotechnology Co., Ltd., Wuhan, China. Then, T7-crRNA-oligo and T7-top oligo were annealed [annealing Buffer for DNA Oligos(5X), Beyotime] to generate a partially dsDNA template, which was finally used for crRNA preparation using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, MA, United States). The crRNA purification was performed using the RNA Clean and Concentrator Kit (New England Biolabs). All crRNAs were examined using a NanoDrop spectrophotometer (Thermo Fisher, Shanghai, China). The DNA oligos for crRNA synthesis are shown in Supplementary Table 2.

In vitro Double-Stranded DNA Substrate Cleavage Assay

To verify the targeted cleavage efficiency of Cas12a combined with different crRNAs, in vitro dsDNA substrate cleavage assay was carried out using the ftsX gene PCR product, LbCas12a (New England Labs), and purified crRNAs. The total volume is 10 μl containing 250 nM LbCas12a, 500 nM crRNA, 1×NEBuffer 2.1 (New England Biolabs), and 5 μl PCR product. The reaction was performed at 37°C for 1 h, and the cleavage products were analyzed by 1% agarose gel electrophoresis.

The ftsX gene PCR amplification was performed in a 25 μl reaction mixture using primer pair XapY17-F and XapY17-R (Tianyi Huiyuan) for 1 μl of each primer (10 μM) (Supplementary Table 3), 1 μl of total DNA (10 ng/μl), 12.5 μl of 2 × Taq PCR MasterMix (YEASEN, Wuhan, China), and 9.5 μl of double-distilled water, which was carried out at 94°C for 4 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 60 s, and a final extension of 72°C for 10 min. The 5 μl PCR products were separated on 1% agarose gels containing gel-red (US Everbright Inc., Suzhou, China) in 1 × TAE buffer and visualized under UV light.

Recombinase Polymerase Amplification Assay

The species-specific ftsX gene was selected to detect Xap. Three pairs of ftsX-specific primers were designed for RPA assay with Primer3¹ according to RPA primer design rules. The primer sequences are available in Supplementary Table 3.

The RPA assay was processed using the TwistAmp Basic Kit (TwistDx, Beijing, China) according to the manufacturer instructions in 50 μl volumes containing 29.5 μl of rehydration buffer, 12.2 μl of nuclease-free H₂O, 480 nM of forward and reverse primers (Tianyi Huiyuan), and 1 μl of genomic DNA (10 ng/μl), which was mixed with freeze-dried RPA enzyme powder. Finally, 2.5 μl of magnesium acetate (280 nM) was added to activate the reaction and incubated at 37°C for 30 min. The RPA products were purified using the DNA Purification Kit (Tianmo Biotech, Beijing, China) and analyzed by 1% agarose gel electrophoresis.

Establishment of the Recombinase Polymerase Amplification/Cas12a-Fluorescence Assay

Generally, an ssDNA probe is needed for the establishment of the RPA/Cas12a analysis to show the presence of the target signal. An FQ-reporter (Tianyi Huiyuan) (Supplementary Table 4) with a FAM fluorophore and a quencher was added to establish the RPA/Cas12a-fluorescence assay, which contained 250 nM LbCas12a, 500 nM crRNA, 1 × NEBuffer 2.1, 500 nM FQ-reporter, and 2–5 μl RPA reaction products in total 10 μl volumes. Reactions were performed at 37°C for 1 h, and the fluorescence intensity was measured on a fluorescence plate reader in the FAM channel. One-way ANOVA with Dunnett’s post-test set at p < 0.05 was used to assess positive results.

For optimizing the concentration ratio of LbCas12a to crRNA, reactions were performed as described above with the following modifications: the LbCas12a concentration was set at 50 nM, the crRNA concentrations were set at 25, 50, 100, 200, and 300 nM for ratio gradients. For optimizing the concentration of LbCas12a, four concentration gradients of 50, 150, 250, and 350 nM were set according to the optimal ratio of LbCas12a to crRNA. The FQ-reporter concentrations were set to 500, 1,000, 1,500, and 2,000 nM for optimal visualization concentration. Furthermore, a mini-UV torch was used to directly visualize the fluorescence signal with the naked eyes.

Finally, the optimized RPA/Cas12a-fluorescence assay was applied to test the sensitivity using a series of concentration gradients from 1 × 10^–11 to 1 × 10^–19 M of the dsDNA targets with three replicates. In addition, two closely related Xanthomonas species/subspecies, nine bacterial species which were collected and identified from peach samples, and four common peach fungal species were examined for assessment of its specificity (Supplementary Table 1).

Establishment of the Recombinase Polymerase Amplification/Cas12a-Lateral Flow Assay

The lateral flow strip was introduced into RPA/Cas12a analysis to resolve restrictions under some situations and improve the applicability. This assay can display the positive band signal at a specified location by binding antibodies and antigens (Supplementary Table 4).

The RPA/Cas12a-LFA assay was carried out according to the optimized conditions of RPA/Cas12a-fluorescence assay. The difference in FQ-reporter was replaced by FB-reporter (Tianyi Huiyuan), the concentration remained 1,000 nM, and other components were unchanged. RPA/Cas12a-LFA reactions were performed at 37°C for 1 h. Finally, 100 μl HybriDetect assay buffer was added and incubated at room temperature for 3 min. Then, the lateral flow strip (TwistDx) was immersed in the reaction tube, and the visible color can be developed in the test band and control band within 2–3 min. The sensitivity and specificity of the RPA/Cas12a-LFA assay were the same as the RPA/Cas12a-fluorescence assays.

Evaluation of the Recombinase Polymerase Amplification/Cas12a Assay With Inoculated and Naturally Infected Samples

Healthy peach leaves were artificially scratched (0.5 cm) and inoculated with 10⁸ CFU/ml Xap bacterial suspension, which was derived from a 2-day-old culture grown in NA liquid culture medium at 28°C. The scratched peach leaves were kept at 28°C for 5–7 days to monitor disease development. The sterile culture solution was used to inoculate the scratched peach leaves as the negative control. A total of five Xap strains were inoculated, and the test was repeated three times. Finally, peach leaf tissues were collected for the NaOH-based DNA crude extraction (3 min), which was directly used for the RPA/Cas12a assay.

Similarly, the naturally infected samples were collected from a peach orchard in Huazhong Agricultural University in Wuhan, Hubei Province, China. They were first identified by ftsX gene PCR and then tested with the RPA/Cas12a assay as described above.

Results

LbCas12a Exhibited Targeted Double-Stranded DNA and Non-targeted Single-Stranded DNA Cleavage Activity

In the beginning, the optimization of RPA parameters including specific primer screening, incubation time, and reaction temperature were performed. It was observed that Xap could be specifically detected by RPA1F/RPA1R primers with high detection efficiency, but other primer sets had all attained weak bands for negative controls. The optimization of reaction conditions only needs to incubate the reactants at 37°C for 15 min (the saturation value) to achieve a fairly high amplification efficiency (Supplementary Figure 1). Then, a total of three crRNAs were designed and synthesized based on the RPA1F/RPA1R targeting sequence for Cas12a cleavage reaction. The schematics of Cas12a-based cleavage reaction using an FQ-reporter were shown in Figure 1A, and the fluorescence signal generated by FQ-reporter degradation indicates the presence of target nucleic acid. The in vitro cleavage reaction illustrated that the crRNA 1, crRNA 2, and crRNA 3 guided LbCas12a had a high cleavage activity for targeted dsDNA, almost attained 100% (Figure 1B). The RPA/Cas12a-based DNA test using an FQ-reporter displayed that the non-targeted ssDNA could also be cleaved by LbCas12a guided by different crRNAs, with the efficacy of crRNA 3 > crRNA 2 > crRNA 1 (Figure 1C). However, in several repeated experiments, we found that the non-targeted ssDNA cleavage activity of LbCas12a guided by crRNA3 was unstable. Therefore, the stable crRNA 2 guided Cas12a-based DNA detection was applied in subsequent experiments.

FIGURE 1

Figure 1. Cas12a-based cleavage reaction combined with recombinase polymerase amplification (RPA) and an FQ-reporter. (A) The schematics of Cas12a-based DNA detection using a FQ-reporter. The starburst indicates fluorophore (e.g., FAM), and the black cycle indicates the quencher of the FQ-reporter. (B) In vitro dsDNA substrate cleavage assay with different crRNAs. M, DNA marker; no-cR, control reaction without crRNA. (C) Detection of the RPA/Cas12a-FQ-reporter assay with different crRNAs. BC, blank control with H₂O.

Establishment of the Recombinase Polymerase Amplification/Cas12a-Fluorescence Assay

First, we set Cas12a at a lower concentration of 50 nM for more obvious distinguishments of the LbCas12a/crRNA ratios optimization. Optimized results indicated that the ratio of LbCas12a to crRNA was negatively correlated with the accumulation of fluorescence signal, and the smaller the ratio, the higher the accumulation of fluorescence signal (Figure 2). Considering that accuracy and reagent cost, the ratio of LbCas12a to crRNA was used as 1:2 (Figure 2A). For the optimization of Cas12a concentration, the fluorescence signal intensity showed a trend of increasing first and then decreasing from 50 to 350 nM, reaching a peak at 150 nM (Figure 2B). Thus, the concentration of LbCas12a was used at 150 nM for the fluorescence assays in subsequent experiments. Furthermore, we also tried to achieve the naked-eye observation of fluorescence signals with a mini-UV torch (Figure 2D). Through optimizing FQ-reporter concentration, it was shown that the green fluorescence signal could be clearly observed when the concentration of FQ-reporter exceeds 1,500 nM, and the best result was observed at 2,000 nM using both a fluorescence plate reader and a mini-UV torch (Figures 2C,E). Thus, the concentration of FQ-reporter was used at 2,000 nM for the fluorescence assay. Finally, the optimized Cas12a system was combined with the optimized RPA to establish the RPA/Cas12a-fluorescence assay.

FIGURE 2

Figure 2. Establishment of the RPA/Cas12a-fluorescence assay. (A) End point of Cas12a-fluorescence assay at different LbCas12a/crRNA ratios. (B) Optimization of LbCas12a concentration. (C) Optimization of FQ-reporter concentration. (D) Visualization with a mini-UV torch. (E) Optimized visualization results of FQ-reporter with a mini-UV torch. The UV light images were taken by the gel imaging analysis system.

Sensitivity and Specificity of the Recombinase Polymerase Amplification/Cas12a-Fluorescence Assay

The ZY2-3-1a gDNA was diluted from 1 × 10^–11 M to 1 × 10^–19 M for sensitivity evaluation. Results showed that the limitation of the RPA/Cas12a-fluorescence assay was 10^–18 M, corresponding to 1.63 copies/μl or 8.855 fg/μl gDNA of Xap (Figures 3A,B), while PCR and RPA assay using the same gDNA had the same detection limitation of 10^–17 M of input, equivalent to 16.3 copies/μl or 88.55 fg/μl (Figure 3C). Therefore, the RPA/Cas12a assay showed better performance than PCR or RPA assay.

FIGURE 3

Figure 3. Sensitivity and specificity of the RPA/Cas12a-fluorescence assay. (A) Sensitivity of the RPA/Cas12a-fluorescence assay. (B) Visualized results of the RPA/Cas12a-fluorescence assay. (C) Sensitivity of the PCR and RPA assay. BC, blank control. (D) (i) Specificity assessment of RPA/Cas12a-fluorescence assay with Xap and bacterial species; (ii) specificity assessment of RPA/Cas12a-fluorescence assay with Xap and other fungal species from the peach; and (iii) visualization of (i) and (ii) with a torch at 365 nm light.

For specificity evaluation, the extracted genomic DNAs of two related Xanthomonas species and subspecies, eight bacterial species from peach leaves, and four common fungal species from peach were tested using the RPA/Cas12a-fluorescence assay. Results showed that Xap could be specifically detected with relatively high fluorescence signal and visualized by the naked eyes, whereas no or low background signal closed to the blank control for the other tested control strains (Figure 3D).

Establishment of the Recombinase Polymerase Amplification/Cas12a-Lateral Flow Assay

The RPA/Cas12a-LFA assay was conducted as shown in Figure 4A, the FQ-reporter was substituted with the FB-reporter, and other reagents remained the same as the RPA/Cas12a-fluorescence assay. The RPA/Cas12a-LFA assay was initially established and tested with Xap, Xaj, and ddH₂O and produced the expected black band in the test line only for Xap (Figure 4B). Similarly, the sensitivity and specificity of the RPA/Cas12a-LFA assay were evaluated in the same way as the RPA/Cas12a-fluorescence assay. The sensitivity of the RPA/Cas12a-LFA assay was tested with 10-fold dilution series (from 1 × 10^–11 M to 1 × 10^–19 M) gDNA. Results showed that the sensitivity reached 10^–17 M, which was 10 times lower than that of the RPA/Cas12a-fluorescence assay (Figure 4C). However, similar to the RPA/Cas12a-fluorescence assay, the RPA/Cas12a-LFA assay also could specifically detect Xap from the other 14 control strains (Figures 4D,E).

FIGURE 4

Figure 4. The RPA/Cas12a-LFA assay. (A) Schematics of RPA/Cas12a-LFA assay. (B) The preliminary establishment of the RPA/Cas12a-LFA system. (C) Sensitivity of the RPA/Cas12a-LFA assay. (D,E) Specificity of the RPA/Cas12a-LFA assay. (F) Field detection of the RPA/Cas12a-LFA assay. BC, blank control.

Field Evaluation of the Simplified Recombinase Polymerase Amplification/Cas12a Assays

Generally, disease diagnosis mainly happens in fields, where complicated equipment cannot be obtained. Therefore, it is crucial to simplify experimental procedures and develop methods, which do not need complicated and expensive equipment. In view of these requirements, we tried to simplify the detection procedure which could be used for simple diagnosis from disease samples directly without DNA purification.

The crude DNA from the NaOH-based extraction methodology was applied to form a simplified RPA/Cas12a detection (Figure 5A). Artificially inoculated and naturally infected peach leaves were directly tested with the simplified RPA/Cas12a detection. Results showed that diseased leaves inoculated with five Xap strains all showed positive results, while peach leaves inoculated with the culture medium did not show any symptoms, and there was no obvious accumulation of fluorescence in the simplified RPA/Cas12a assays (Figure 5B). Similarly, the analogous fluorescence was only observed in the infected samples when natural peach leaves were investigated (Figure 5C).

FIGURE 5

Figure 5. Field evaluation of the RPA/Cas12a-fluorescence assay. (A) Schematic of the integrated RPA/Cas12a analysis. (B) The RPA/Cas12a-fluorescence detection of inoculated leaves. (C) The RPA/Cas12a-fluorescence detection of natural leaves. BC, blank control.

To further test the feasibility of the simplified RPA/Cas12a assay, the 37 natural peach leaf samples were collected and tested from an orchard in Huazhong Agricultural University. As expected, the RPA/Cas12a-fluorescence assay exhibited consistent results with ftsX gene PCR, 3 positive samples, and 34 negative samples (Figure 6). Then, three positive samples and five randomly selected negative samples were picked for simultaneous verification using the simplified RPA/Cas12a-LFA assay, and consistent results were observed (Figure 4F). Consequently, the simplified RPA/Cas12a assay was practicable for Xap diagnosis and monitoring of bacterial spot disease.

FIGURE 6

Figure 6. Field detection of natural peach leaves with the simplified RPA/Cas12a-fluorescence assay. (A) A total of 37 peach leaf samples were investigated with PCR amplification. (B) The simplified RPA/Cas12a-fluorescence assay was applied for the same 37 leaf samples.

Discussion

In recent years, the peach bacterial spot has caused huge economic losses in China. This disease occurred as a rapid outbreak across the nation, especially in the middle reach of the Yangtze River where there is long-term rainfall and high humidity. Considering its severity and explosiveness, accurate and fast diagnosis of the peach bacterial spot is important and urgent for surveillance and control of its outbreaks. However, current diagnostic technology often requires laboratory facilities and delivery of samples to a qualified laboratory, which delays the progress of diagnosis and increases the probability of a pandemic. Therefore, it is necessary to innovate a simple, fast, and efficient tool, which can not only realize rapid and fast diagnosis under field conditions but also maintain high accuracy.

Recently, CRISPR/Cas-based diagnostics is emerging as a promising technology for nucleic acid detection owing to its simplicity and sensitivity (Zhang et al., 2020). Obviously, a variety of applications have been derived, such as the diagnosis of African swine fever (Wang et al., 2020), HPV16 and HPV18, the causal agent of cervical cancer (Mukama et al., 2020), new coronavirus disease 2019, etc. (Broughton et al., 2020). In addition, related applications in plants have also been proposed. For instance, Cas12a is engineered to detect the virus of grapevine and CaMV35S promoter in genetically modified soybeans (Li et al., 2019b; Wu et al., 2019). Zhang et al. (2020) first utilized the CRISPR/Cas12a system to realize the fast detection of the rice blast pathogen and GMO in rice. In view of these, we can anticipate that this Cas12a-based DNA detection method could be broadly used in disease diagnosis with some modifications.

The crRNA is critically important for Cas12a-based detection. In this study, a total of three crRNAs were designed for the Cas12a cleavage reaction. Surprisingly, all of them successfully degraded the dsDNA target in vitro. We guessed that this may rely on CRISPR/Cas12a (Cpf1) which uses a single RuvC catalytic domain for guide RNA-directed dsDNA cleavage. The compatibility of crRNA and dsDNA affects the stability of Cas12a cleavage conformation, which determines products with different lengths (Zhang et al., 2019). LbCas12a exhibited the non-target ssDNA cleavage reaction which was revealed using an FQ-reporter, and all crRNAs appeared obvious fluorescence signal accumulation, while signal intensities vary frequently, such as crRNA3. It is possible because the targeting sequence of crRNA plays a key role to determine the editing efficiency in Cas12a-based genome editing (Kim et al., 2017). Finally, we chose a stable crRNA2 for subsequent innovations. This fully illustrates the necessity of designing multiple crRNAs simultaneously for the Cas12a-based test against specific targets.

Based on a series of experiments, we developed the RPA/Cas12a assays for peach Xap detection. We further simplified the RPA/Cas12a analysis with the crude DNA from peach leaves directly, which realized the Xap detection at body temperature within 2 h. Actually, this is the first report based on RPA or Cas12a system for Xap detection. We provided two visualization approaches with a mini-UV torch or immunochromatographic lateral flow strips so that the operators can adjust the experimental scheme freely according to the available conditions on-site, which greatly enhanced the detection practicability and resolved some bottlenecks that could not be conducted previously under field conditions. This new assay comprises two steps, RPA reaction first recognizes and amplifies target signals, and Cas12a-based dsDNA targets and non-target ssDNA cleavage reaction forms the secondary detection which effectively avoids false-positive errors and ensures high accuracy. Unlike other technologies, the simplified RPA/Cas12a assays do not require complex instruments, only 37°C incubation for 1 h, which can be easily attained by the body temperature under field conditions. Furthermore, the RPA/Cas12a assay maintains high sensitivity of more than 10^–17 M of Xap gDNA and high specificity which could accurately distinguish the Xap from other peach pathogens, even from the closely related pathovar Xaj in the same species X. arboricola. Considering that the whole test procedure is easy, rapid, flexible, and effective, peach growers or other users with basic knowledge can easily carry out the test and analyze the result under field conditions. We have full confidence that this technology will soon reach the greatest degree of universal application in practice. To further enhance the usage of this technology in future, corresponding detection kits should be produced.

Certainly, there are also some potential limitations and future improvements that should be noted. First, in the specificity of the RPA/Cas12a-fluorescence assay or the RPA/Cas12a-LFA assay, the background fluorescence signal is occasionally too high which may cause false-positive results. In this case, reducing the concentration of the FQ-reporter might be a way to solve this issue. Although it is still unclear, some reasonable conjectures could be raised. Mukama et al. (2020) has shown that the length and sequence of FQ-reporter affect the non-specific ssDNA cleavage activity of Cas12a. The RNA-independent nuclease activities and off-targeting effect of Cas12a-based genome editing may be related to this, leading to some false positive situations. In addition, the aerosol pollution caused by RPA amplification may lead to some background signals. Second, RPA targeting sequences should be rich in multiple PAM sites so that multiple crRNAs can be designed for Cas12a cleavage reaction. Finally, based on the observation of the fluorescence curve, it only took 30 min to achieve a fairly high fluorescence accumulation, and the reaction gradually reached a plateau. The field evaluation of the RPA/Cas12a assay illustrated that obvious fluorescence signals could also be observed within 30 min. However, the shorter cleavage time may cause false-negative results in the detection of low biomass DNA samples, and the detection sensitivity may be significantly reduced. Actually, the sensitivity of diagnosis based on the LFA strip can be significantly reduced to 10^–13M when the reaction time is reduced to 30 min (Supplementary Figure 2). To obtain accurate results, we suggested that the Cas12a cleavage reaction can be carried out for 1 h. In view of this, the NaOH-based DNA crude extraction should be further optimized to increase the DNA yield and purity so that Xap can be detected even at the early stage of the low bacterial population. Besides, we can appropriately shorten the reaction time to 30 min to improve detection efficiency wherever good sample quality is available.

Conclusion

We developed an easy, fast, and effective assay for Xap detection. It could achieve rapid detection of Xap at 37°C within 2 h, and two flexible approaches have been designed for users to choose. We also launched the simplified RPA/Cas12a assay program, which can realize batch sample detection directly under field conditions. Based on the advantages and advancements, we believed that this RPA/Cas12a assay will soon be introduced into commercial use in practice.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Author Contributions

C-XL conceived the idea and finalized the manuscript. C-XL and ML designed the experiments. ML, F-ZM, and QT performed the experiments and analyzed the data. ML, W-XY, and C-XL wrote the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This study was supported by the China Agriculture Research System of MOF and MARA and the Fundamental Research Funds for the Central Universities (No. 2662020ZKPY018).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor declared a past collaboration with one of the authors W-XY.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

This is a short text to acknowledge the contributions of specific colleagues, institutions, or agencies that aided the efforts of the authors. We thank K. B. Xie for his helpful, critical technical assistance with the RPA/Cas12a system.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021.740177/full#supplementary-material

Footnotes

1. ^ http://bioinfo.ut.ee/primer3/

References

Bai, J., Lin, H., Lin, H., Zhou, Y., Liu, J., Zhong, G., et al. (2019). Cas12a-based on-site and rapid nucleic acid detection of African swine fever. Front. Microbiol. 10:2830. doi: 10.3389/fmicb.2019.02830

PubMed Abstract | CrossRef Full Text | Google Scholar

Bock, C. H., Hotchkiss, M. W., Okie, W. R., and Wood, B. W. (2011). The distribution of peach scab lesions on the surface of diseased peaches. Eur. J. Plant Pathol. 130, 393–402. doi: 10.1007/s10658-011-9761-y

CrossRef Full Text | Google Scholar

Broughton, J. P., Deng, X., Yu, G., Fasching, C. L., and Chiu, C. Y. (2020). CRISPR-Cas12-based detection of SARS-COV-2. Nat. Biotechnol. 8, 870–874. doi: 10.1038/s41587-020-0513-4

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., et al. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439. doi: 10.1126/science.aar6245

PubMed Abstract | CrossRef Full Text | Google Scholar

European and Mediterranean Plant Protection Organization [EPPO] (2006). Standards PM 7/64. Diagnostics protocols for regulated pests. Xanthomonas arboricola pv. pruni. Bull. OEPP EPPO Bull. 36, 129–133.

Google Scholar

Gootenberg, J. S., Abudayyeh, O. O., Kellner, M. J., Joung, J., Collins, J. J., and Zhang, F. (2018). Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360, 439–444. doi: 10.1126/science.aaq0179

PubMed Abstract | CrossRef Full Text | Google Scholar

Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., et al. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442. doi: 10.1126/science.aam9321

PubMed Abstract | CrossRef Full Text | Google Scholar

Harrington, L. B., Burstein, D., Chen, J. S., Paez-Espino, D., Ma, E., Witte, I. P., et al. (2018). Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science 362, 839–842. doi: 10.1126/science.aav4294

PubMed Abstract | CrossRef Full Text | Google Scholar

Janse, J. D. (2012). Bacterial diseases that may or do emerge, with (possible) economic damage for Europe and the Mediterranean basin: Notes on epidemiology, risks, prevention and management on first occurrence. J. Plant Pathol. 94, 5–29.

Google Scholar

Kim, H. K., Song, M., Lee, J., Menon, A. V., Jung, S., Kang, Y. M., et al. (2017). In vivo high-throughput profiling of CRISPR-Cpf1 activity. Nat. Methods 14, 153–159. doi: 10.1038/nmeth.4104

PubMed Abstract | CrossRef Full Text | Google Scholar

Knott, G. J., and Doudna, J. A. (2018). CRISPR-Cas guides the future of genetic engineering. Science 361, 866–869. doi: 10.1126/science.aat5011

PubMed Abstract | CrossRef Full Text | Google Scholar

Li, S. Y., Cheng, Q. X., Wang, J. M., Li, X. Y., Zhang, Z. L., Gao, S., et al. (2018). CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 4:20. doi: 10.1038/s41421-018-0028-z

PubMed Abstract | CrossRef Full Text | Google Scholar

Li, Y., Li, S., Wang, J., and Liu, G. (2019a). CRISPR/Cas systems towards next-generation biosensing. Trends Biotechnol. 37, 730–743. doi: 10.1016/j.tibtech.2018.12.005

PubMed Abstract | CrossRef Full Text | Google Scholar

Li, Y., Mansour, H., Wang, T., Poojari, S., and Li, F. (2019b). Naked-eye detection of grapevine red-blotch viral infection using a Plasmonic CRISPR Cas12a assay. Anal. Chem. 91, 11510–11513. doi: 10.1021/acs.analchem.9b03545

PubMed Abstract | CrossRef Full Text | Google Scholar

Mukama, O., Yuan, T., He, Z., Li, Z., and Zeng, L. (2020). A high fidelity Crispr/Cas12a based lateral flow biosensor for the detection of hpv16 and hpv18. Sensor Actuat. B Chem. 316, 119–128. doi: 10.1016/j.snb.2020.128119

CrossRef Full Text | Google Scholar

Myhrvold, C., Freije, C. A., Gootenberg, J. S., Abudayyeh, O. O., Metsky, H. C., Durbin, A. F., et al. (2018). Field-deployable viral diagnostics using CRISPR-Cas13. Science 360, 444–448. doi: 10.1126/science.aas8836

PubMed Abstract | CrossRef Full Text | Google Scholar

Oura, C. A. L., Edwards, L., and Batten, C. A. (2013). Virological diagnosis of African Swine Fever-comparative study of available tests. Virus Res. 173, 150–158. doi: 10.1016/j.virusres.2012.10.022

PubMed Abstract | CrossRef Full Text | Google Scholar

Palacio-Bielsa, A., Cubero, J., Cambra, M. A., Collados, R., and López, M. M. (2011). Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in prunus species. Appl. Environ. Microb. 77, 89–97. doi: 10.1128/AEM.01593-10

PubMed Abstract | CrossRef Full Text | Google Scholar

Park, S. Y., Lee, Y. S., Koh, Y. J., Hur, J. S., and Jung, J. S. (2010). Detection of Xanthomonas arboricola pv. pruni by PCR using primers based on DNA sequences related to the hrp genes. J. Microbiol. 48, 554–558. doi: 10.1007/s12275-010-0072-3

PubMed Abstract | CrossRef Full Text | Google Scholar

Piepenburg, O., Williams, C. H., Stemple, D. L., and Armes, N. A. (2006). DNA detection using recombination proteins. PLoS Biol. 4:e204. doi: 10.1371/journal.pbio.0040204

PubMed Abstract | CrossRef Full Text | Google Scholar

Pothier, J. F., Pagani, M. C., Pelludat, C., Ritchie, D. F., and Duffy, B. (2011a). A duplex-PCR method for species and pathovar level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni. J. Microbiol. Meth. 86, 16–24. doi: 10.1016/j.mimet.2011.03.019

PubMed Abstract | CrossRef Full Text | Google Scholar

Pothier, J. F., Vorhölter, F. J., Blom, J., Goesmann, A., Pühler, A., Smits, T. H., et al. (2011b). The ubiquitous plasmid pxap41 in the invasive phytopathogen Xanthomonas arboricola pv. pruni: complete sequence and comparative genomic analysis. FEMS Microbiol. Lett. 323, 52–60. doi: 10.1111/j.1574-6968.2011.02352.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Qian, W. J., Lu, Y., Meng, Y., Ye, Z., Wang, L., Wang, R., et al. (2018). Field detection of citrus huanglongbing associated with ‘Candidatus liberibacter asiaticus’ by recombinese polymerase amplification within 15 min. J. Agr. Food Chem. 66, 5473–5480. doi: 10.1021/acs.jafc.8b01015

PubMed Abstract | CrossRef Full Text | Google Scholar

Rosello, M., Santiago, R., Palacio-Bielsa, A., García-Figueres, F., Montón, C., Cambra, M. A., et al. (2012). Current status of bacterial spot of stone fruits and almond caused by Xanthomonas arboricola pv. pruni in Spain. J. Plant Pathol. 94, 15–21. doi: 10.4454/jpp.v94i1sup.004

PubMed Abstract | CrossRef Full Text | Google Scholar

Sashital, D. G. (2018). Pathogen detection in the CRISPR-Cas era. Genome Med. 10:32. doi: 10.1186/s13073-018-0543-4

PubMed Abstract | CrossRef Full Text | Google Scholar

Stefani, E. (2010). Economic significance and control of bacterial spot/canker of stone fruits caused by Xanthomonas arboricola pv. pruni. J. Plant Pathol. 92, 99–103.

Google Scholar

Suleman, E., Mtshali, M. S., and Lane, E. (2016). Investigation of false positives associated with loop-mediated isothermal amplification assays for detection of toxoplasma gondii in archived tissue samples of captive felids. J. Vet. Diagn. Investig. 28, 536–542. doi: 10.1177/1040638716659864

PubMed Abstract | CrossRef Full Text | Google Scholar

Teng, F., Guo, L., Cui, T., Wang, X. G., Xu, K., Gao, Q., et al. (2019). CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity. Genome Biol. 20:132. doi: 10.1186/s13059-019-1742-z

PubMed Abstract | CrossRef Full Text | Google Scholar

Tjou-Tam-Sin, N. N. A., van de Bilt, J. L. J., Bergsma-Vlami, M., Koenraadt, H., Naktuinbouw, J. W., van Doorn, J., et al. (2014). First report of Xanthomonas arboricola pv. pruni in ornamental Prunus laurocerasus in the Netherlands. Plant Dis. 96:759.

Google Scholar

Wang, X., Ji, P., Fan, H., Dang, L., and Liao, M. (2020). CRISPR/Cas12a technology combined with immunochromatographic strips for portable detection of african swine fever virus. Commun. Biol. 3:62. doi: 10.1038/s42003-020-0796-5

PubMed Abstract | CrossRef Full Text | Google Scholar

Wu, H., He, J. S., Zhang, F., Ping, J., and Wu, J. (2019). Contamination-free visual detection of CaMV35S promoter amplicon using CRISPR/Cas12a coupled with a designed reaction vessel: rapid, specific and sensitive. Anal. Chim. Acta 1096, 130–137.

Google Scholar

Zhang, L. J., Sun, R., Yang, M., Peng, S., Cheng, Y., Chen, C., et al. (2019). Conformational dynamics and cleavage sites of Cas12a are modulated by complementarity between crRNA and DNA. Science 19, 492–503. doi: 10.1016/j.isci.2019.08.005

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhang, Y. M., Zhang, Y., and Xie, K. (2020). Evaluation of CRISPR/Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice. Mol. Breeding. 40:11. doi: 10.1007/s11032-019-1092-2

CrossRef Full Text | Google Scholar