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Rapid and visual RPA-CRISPR/Cas12a detection for Staphylococcus pseudintermedius and methicillin-resistant S. pseudintermedius in clinical samples of dogs and cats

One Health Advances volume 1, Article number: 20 (2023) Cite this article

Abstract

Staphylococcus pseudintermedius can cause severe infections of the skin, ear and other tissues in dogs and cats. Methicillin-resistant S. pseudintermedius (MRSP) has recently become more prevalent, posing a severe threat to companion animals and public health. Therefore, rapid and accurate diagnosis of S. pseudintermedius and MRSP infections in dogs and cats is essential for timely controlling infections. The development of CRISPR/Cas technology offers an innovative solution for rapid diagnosis. Here, we established an assay combining recombinant polymerase amplification (RPA) and CRISPR/Cas12a. By separately detecting the spsJ gene, the specific gene of S. pseudintermedius, and the mecA gene, the methicillin resistance gene, this method allows for the direct detection of methicillin-susceptible S. pseudintermedius (MSSP) and MRSP in clinical samples at 37 °C for a total of 40 min, The results can be directly visualized by the naked eye under blue light. The limits of detection of the RPA-CRISPR/Cas12a assay were 103 copies per reaction for the spsJ gene and 104 copies per reaction for the mecA gene. The RPA-CRISPR/Cas12a detection successfully detected and differentiated clinical isolates of MSSP and MRSP without cross-reactivity with other tested bacteria species. The evaluation of the detection performance of RPA-CRISPR/Cas12a with 47 clinical samples (without culture) from dogs and cats showed that the results of detection were 100% consistent with those of clinical culture and colony sequencing, which was more sensitive than PCR. RPA-CRISPR/Cas12a assay can quickly and sensitively detect S. pseudintermedius and MRSP in clinical samples without expensive instruments, making it suitable for small veterinary clinics.

Introduction

Staphylococcus pseudintermedius, coagulase-positive Staphylococcus, is a commensal of the skin and mucosa of dogs and cats, and an opportunistic pathogen [1]. It is the most frequent bacterial pathogen isolated from clinical canine samples, mainly associated with skin and ear infections [2], and can also cause other infections such as wound infections, lung infections, urinary tract infections and osteomyelitis [3]. S. pseudintermedius is not a normal colonizer of human skin and mucous, however, it may colonize in humans who are in close contact with dogs. Especially when human immune system is compromised, it may cause disease [4,5,6]. S. pseudintermedius infections in people have been associated with bloodstream infections, abscesses, pneumonia, and septic arthritis, according to research [7,8,9].

Antibiotic resistance is a significant issue for S. pseudintermedius due to the extensive use of antibiotics in canine and feline infections. Methicillin-resistant S. pseudintermedius (MRSP) is one of the most commonly reported drug-resistant strains. MRSP was initially reported in Europe in 2006 [10]. In recent years, there has been a global increase in the prevalence of MRSP infection [11]. Reports indicated that 5.8 ~ 32.2% of S. pseudintermedius isolated from dogs and cats were methicillin-resistant [12]. Methicillin resistance of MRSP is mediated by the mecA gene, which encodes a modified penicillin-binding protein 2a (PBP2a). PBP2a has low affinity to most β-lactam antibiotics [13], resulting in MRSP resistance to almost all β-lactams except ceftaroline and ceftobiprole [14]. MRSP challenges the use of antibiotics in dogs and cats since β-lactam antibiotics are the first line of defense against staphylococcal infections [15]. The mecA gene exists on the staphylococcal chromosomal cassette mec (SCCmec), a mobile element of the bacterial chromosome. SCCmec can be transferred between different Staphylococcus species, leading to the spread of drug resistance [16]. Therefore, rapid and sensitive detection of S. pseudintermedius and MRSP is critical for early diagnosis of S. pseudintermedius infections in dogs and cats and the rational use of antibiotics.

In veterinary clinics, S. pseudintermedius is usually isolated and identified through phenotype [2]. The antibacterial susceptibility test is detected by oxacillin disc diffusion or broth microdilution [17]. However, these assays are time-consuming (2–3 days), cumbersome, and susceptible to culture conditions. Other molecular-based methods, such as PCR (polymerase chain reaction) [18], multiplex-PCR [19], qPCR (real-time quantitative PCR) [20], and PCR-restriction fragment length polymorphism [21] can directly detect the specific genes of S. pseudintermedius and mecA genes. These methods can significantly improve detection sensitivity but require expensive equipment and trained personnel. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) provides rapid and highly accurate identification of S. pseudintermedius [22, 23]. However, the very high cost of the MALDI-TOF instrument limits its use.

Recently, the discovery of the CRISPR (cluster regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system has offered an innovative solution for rapid diagnosis. The CRISPR system targets DNA guided by RNA [24]. The Cas12a-crRNA (CRISPR RNA) complex specifically recognizes the target DNA and activates the trans-cleavage activity of Cas12a to cut single-stranded DNA (ssDNA) indiscriminately [25]. Genetic detection can be performed by designing crRNAs for different target genes and activating trans-cleavage activity of Cas12a to cut ssDNA [26]. Several CRISPR/Cas12a-based genetic assays have been developed, including DETECR (DNA endonuclease-targeted CRISPR trans reporter) [27] and HOLMES (one-hour low-cost multipurpose highly efficient system) [28]. When CRISPR/Cas12a is combined with isothermal amplification techniques such as recombinase polymerase amplification (RPA) [25, 29] or loop-mediated isothermal amplification (LAMP) [30,31,32], pathogenic bacteria can be detected in a sensitive and specific manner.

Here, we established the RPA-CRISPR/Cas12a (RPA combined with CRISPR/Cas12a) detection method that can directly, accurately, and sensitively detect S. pseudintermedius and MRSP in clinical samples. We selected the S. pseudintermedius surface gene (spsJ gene) for the specific detection of S. pseudintermedius [20, 33] and the mecA gene for the specific detection of methicillin resistance. The detection process and principle of RPA-CRISPR/Cas12a were shown in Fig. 1. The RPA-CRISPR/Cas12a detection for S. pseudintermedius and MRSP was important for rapid and accurate diagnosis of S. pseudintermedius infection and guiding the rational use of antibiotics.

Fig. 1
figure 1

The workflow of RPA-CRISPR/Cas12a detection of S. pseudintermedius and MRSP in clinical samples. Genomic DNA was quickly extracted from clinical samples of dogs and cats using DNA extraction buffer. The spsJ gene and mecA genes were amplified by RPA, and the products were transferred to the CRISPR/Cas12a detection system comprising specific crRNAs targeting the spsJ and mecA genes. The Cas12a-crRNA complex bound to the target DNA activated the trans-cleavage activity of Cas12a and cleaved the ssDNA reporter labeled with FAM and BHQ1 at either end. The product produced an intense fluorescence signal at a specific wavelength. The fluorescence can be detected by a fluorescence microplate reader or observed directly by the naked eye under blue light as green fluorescence. The diagnosis was MRSP when both spsJ and mecA genes were positive; MSSP when spsJ gene was positive but mecA gene was negative. The diagnosis was S. pseudintermedius negative and other mecA positive Staphylococcus when spsJ was negative but mecA was positive. The diagnosis was S. pseudintermedius negative when both spsJ and mecA were negative. The entire reaction can be finished in 40 min at 37 °C

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Results

Establishment and optimization of RPA reaction system

Two sets of forward and reverse primers were initially designed for the spsJ and mecA genes, respectively. When the primer pairs RPA-spsJ-F1/R2 (spsJ gene) and RPA-mecA-F2/R2 (mecA gene) participated in RPA using plasmids DNA containing spsJ or mecA as template, the amplified products had the strongest bands with less non-specific amplification (Fig. 2A, D). Therefore, these primer pairs were selected as the optimal primers for RPA. To determine the best reaction temperature, RPA was performed at 37 °C, 39 °C, and 42 °C using the best primer pairs. The bands of the amplified products were all quite strong regardless of the difference in temperature (Fig. 2B, E). Since the CRISPR/Cas12a reaction temperature was 37 °C and there was no need to change the temperature between the RPA and CRISPR/Cas12a reactions, we decided to use that temperature for the RPA reaction. To evaluate the effect of reaction time on RPA amplification yield, RPA was performed for 10, 15, 20, and 25 min, respectively. Figure 2C and F showed that increasing reaction time led to higher yield of amplified products. RPA products of different times were involved in the CRISPR/Cas12a reaction. It was observed that when RPA was performed for 15 min, it triggered high-intensity endpoint fluorescence in the CRISPR/Cas12a reaction (Fig. S1). To shorten the overall reaction time, 15 min was chosen as the amplification time for RPA.

Fig. 2
figure 2

Optimization of RPA reaction conditions. Optimization of RPA primer pairs (A), reaction temperature (B), and reaction time (C) for detection of the spsJ gene. Optimization of RPA primer pairs (D), reaction temperature (E), and reaction time (F) for detection of the mecA gene. 104 copies of the spsJ plasmid (containing part of the spsJ gene) and 104 copies of the mecA plasmid (containing part of the mecA gene) were used as templates for RPA. M, marker. NTC, no-target control

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Optimization of CRISPR/Cas12a reaction system and establishment of RPA-CRISPR/Cas12a detection

The CRISPR/Cas12a reaction system relies on crRNA to guide Cas12a cleavage efficiency. The crRNA sequence was screened, and the concentration of crRNA and Cas12a was optimized. Three crRNAs were designed for the spsJ and mecA genes, respectively, and the crRNAs were employed in the CRISPR/Cas12a reaction. Figures 3A, B showed that crRNA-spsJ2 and crRNA-mecA3 had the best efficiency in the CRISPR/Cas12a reaction with the highest fluorescence intensity at the same time. The concentration of crRNA and Cas12a was then optimized. The highest endpoint fluorescence intensity was observed at crRNA concentration of 2 μM (Fig. 3C, D), and Cas12a concentration of 30 nM (Fig. 3E, F).

Fig. 3
figure 3

Optimization of the Cas12a reaction system. Optimization of the crRNA for the spsJ gene (A) and the mecA gene (B). Optimization of crRNA concentration for the spsJ gene (C) and the mecA gene (D). Optimization of Cas12a concentration for the spsJ gene (E) and the mecA gene (F). The purified product of PCR of spsJ plasmid or mecA plasmid was used as the substrate of Cas12a reaction, and the concentration of substrate was 6.25 nM. NTC, no-target control. Data were represented as mean ± standard deviation (SD) (n = 3 technical replicates). All data were compared with NTC for significant difference. **** p < 0.0001; *** p < 0.001

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To establish the RPA-CRISPR/Cas12a detection system, the RPA-CRISPR/Cas12a reaction was performed under the optimal RPA and Cas12a reaction conditions using spsJ or mecA plasmids as templates, respectively. In Fig. 4A, B, the products produced obvious fluorescence signals, emitting green fluorescence under blue light in the presence of target genes. The fluorescence intensity did not increase significantly in absence of the target gene, and there was no obvious fluorescence visible to the naked eye (Fig. 4B). This indicated that we have successfully established the RPA-CRISPR/Cas12a method for detecting the spsJ gene and mecA gene. It was evaluated that when the ratio of the endpoint fluorescence intensity of the sample to be tested to that of the negative control was greater than two (S/N > 2), significant fluorescence of the sample to be tested could be observed by the naked eye, and the fluorescence intensity differed significantly from that of the negative control.

Fig. 4
figure 4

Establishment of RPA-CRISPR/Cas12a detection. Real-time fluorescence intensity (A) and endpoint fluorescence intensity (B) of the spsJ and mecA genes were detected by RPA-CRISPR/Cas12a. NTC, no-target-control. Data were expressed as mean ± SD (n = 3 technical replicates). All data were compared with NTC for significant difference. **** p < 0.0001

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Sensitivity of RPA-CRISPR/Cas12a detection

The sensitivity of the RPA-CRISPR/Cas12a detection system was evaluated using spsJ and mecA plasmid DNA (106–100 copies / μL) as templates and compared with PCR. As shown in Fig. 5A, for spsJ, the detection can detect at least 103 copies of spsJ plasmids. For mecA, the system can detect 104 copies of mecA plasmids (Fig. 5B). Therefore, for the spsJ gene and mecA gene, the limits of detection (LOD) of RPA-CRISPR/Cas12a were 103 copies and 104 copies, respectively. For PCR, the bands were obvious when there were 105 copies of plasmids in the reaction system, but they were difficult to see when there were 104 copies (Fig. 5C, D). The LOD of PCR was 105 copies per reaction, indicating that the RPA-CRISPR/Cas12a detection was more sensitive.

Fig. 5
figure 5

Sensitivity evaluation of RPA-CRISPR/Cas12a detection and PCR. Sensitivity evaluation of RPA-CRISPR/Cas12a detection of spsJ gene (A) and mecA gene (B). Sensitivity evaluation of PCR of spsJ gene (C) and mecA gene (D). The serial 10-fold dilution of spsJ and mecA plasmids (106–100 or 108–100 copies/reaction) was used as templates to evaluate the sensitivity of RPA-CRISPR/Cas12a detection and PCR. The sensitivity evaluation results of RPA-CRISPR/Cas12a detection were shown by the endpoint fluorescence intensity and endpoint fluorescence photography. The sensitivity of PCR was shown by agarose gel electrophoresis. M, marker. NTC, no-target control. Data were expressed as mean ± SD (n = 3 technical replicates). All data were compared with NTC for significant difference. **** p < 0.0001; ns, not significant

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Specificity of RPA-CRISPR/Cas12a detection

To evaluate the specificity of RPA-CRISPR/Cas12a for detecting spsJ and mecA genes, 10 MRSP strains, 10 MSSP strains, 6 Staphylococcus strains of non-S. pseudintermedius and 7 non-Staphylococcus strains were used in the reaction. The specific information of the strains was listed in Table S1. Only when S. pseudintermedius participated in the reaction did the fluorescence intensity of the RPA-CRISPR/Cas12a reaction for detecting the spsJ gene increase significantly and did not cross-react with other species (Fig. 6A). Similarly, only when Staphylococcus carrying the mecA gene participated in the reaction did the fluorescence intensity of RPA-CRISPR/Cas12a reaction for detecting the mecA gene increase significantly (Fig. 6B). These results showed that the RPA-CRISPR/Cas12a detection had great specificity, no cross-reactivity, and could diagnose spsJ gene and mecA gene specifically. This method allowed specific detection of MRSP and MSSP.

Fig. 6
figure 6

Specificity evaluation of the RPA-CRISPR/Cas12a detection. The endpoint fluorescence intensity and endpoint fluorescence photography of the RPA-CRISR/Cas12a reaction for the detection of the spsJ gene (A) and the mecA gene (B). NTC, no-target control. Data were expressed as mean ± SD (n = 3 technical replicates)

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Detection of clinical samples

To evaluate the effectiveness of the RPA-CRISPR/Cas12a detection system in clinical samples, forty-seven clinical samples of dogs and cats were collected, including 21 skin samples (n = 21) and 26 ear canal samples (n = 26). The clinical samples were cultured, and PCR was performed on DNA extracted from cultured colonies to identify the spsJ and mecA genes and the products were sequenced, and this result was taken as the "gold standard". Among all the clinical samples collected, MRSP was isolated and identified from samples No. 1-27, MSSP was isolated only from samples No. 28-40, and S. pseudintermedius and the mecA gene were not isolated from samples No. 41-47 (Table S2). The RPA-CRISPR/Cas12a assay and direct PCR were used in the crude genomic DNA of clinical samples. When RPA-CRISPR/Cas12a was used to detect the spsJ gene, the fluorescence intensity of clinical samples No. 1-40 matched our predefined standard of positive results of S/N > 2. When detecting the mecA gene, the fluorescence intensity of clinical samples No. 1-27 matched the standard of positive results of S/N > 2, and other tests matched the standard of negative results of S/N < 2. As a result, RPA-CRISPR/Cas12a correctly detected 27 MRSP samples, 13 MSSP samples, and 7 S. pseudintermedius negative samples (Fig. 7A, B). Direct PCR correctly detected 25 MRSP clinical samples, 13 MSSP samples, and 7 S. pseudintermedius negative samples, MRSP was incorrectly identified as MSSP in samples 2 and 23 (Fig. S2). The coincidence rate between RPA-CRISPR/Cas12a test results and gold standard was 100%, while the coincidence rate of direct PCR with gold standard was 95.7% (Table 1).

Fig. 7
figure 7

Application of RPA-CRISPR/Cas12a detection in 47 clinical samples. (A) Endpoint fluorescence intensity of the spsJ gene and mecA gene in clinical samples was detected separately employing RPA-CRISPR/Cas12a detection. Different shades of rad indicated positive results, and white indicated negative results. (B) Endpoint fluorescence photography of the spsJ and mecA genes in clinical samples employing RPA-CRISPR/Cas12a detection. NTC, no-target control

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Table 1 Evaluation of RPA-CRISPR/Cas12a detection for detecting spsJ and mecA genes in clinical samples
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Discussion

S. pseudintermedius can cause serious infections of the skin, ear, and other tissues. It is the primary cause of canine pyoderma, which can also lead to otitis externa, urinary tract infections, and respiratory infections [3]. Due to the inappropriate use of antibiotics and the lack of new antibiotics development, the rate of methicillin resistance in S. pseudintermedius has rapidly increased [34], which poses a challenge to the use of antibiotics. Therefore, rapid and reliable diagnosis of S. pseudintermedius and MRSP is critical for early diagnosis and treatment of diseases. Traditional methods for diagnosing S. pseudintermedius and drug resistance based on the isolation and culture of bacteria and antibacterial susceptibility test are time-consuming, cumbersome, and unsuitable for rapid clinical diagnosis. Although PCR-based methods can rapidly and sensitively diagnose specific genes and methicillin resistance genes of S. pseudintermedius, they are not suited for on-site detection since they rely on laboratory conditions, equipment, and trained personnel.

The RPA used in this study is an isothermal amplification technique that uses the activity of biological enzymes to amplify nucleic acids at isothermal conditions of 37 ~ 42 °C without complex instrumentation [35]. The target amplification products can be obtained within 5 ~ 20 min, allowing portable and rapid nucleic acid detection. However, RPA can tolerate mismatches, primers and templates can mismatch up to nine base pairs [36,37,38], causing non-specific amplification and false positive in RPA which lower the accuracy of the results. CRISPR/Cas12a detection has high specificity provided by the complementarity between crRNA and target DNA. When there is a base mismatch between crRNA and target DNA, especially in the seed region, the cleavage activity of Cas12a will be significantly reduced [24, 39]. The specificity of the detection can be significantly increased by combining RPA with CRISPR/Cas12a. When we investigated the specificity of detection of S. pseudintermedius using RPA-CRISPR/Cas12a, we found that when using RPA alone, the RPA products of Staphylococcus other than S. pseudintermedius also showed non-specific bands after agarose gel electrophoresis, but when using CRISPR/Cas12a to detect the RPA products, only S. pseudintermedius amplification products produced bright green fluorescence, indicating the high specificity of our RPA-CRISPR/Cas12a detection method.

In this study, we developed a rapid and accurate method for detecting S. pseudintermedius and MRSP. This method was more sensitive than PCR and provided visual results at a constant temperature of 37 °C for 40 min without the use of expensive instruments. The RPA-CRISPR/Cas12a had LOD of 103 copies for the spsJ gene and 104 copies for the mecA gene, which was higher than that of PCR at 105 copies. We verified its high specificity to specifically identify MRSP and MSSP strains by testing Staphylococcus strains and non-Staphylococcus strains. Additionally, we verified its capacity to identify S. pseudintermedius and MRSP in the crude genomic DNA of clinical samples. The coincidence rate between the results of RPA-CRISPR/Cas12a detection in 47 clinical samples and the gold standard was 100%, while that of PCR was 95.7%. These results indicated that RPA-CRISPR/Cas12a can more sensitively and specifically detect S. pseudintermedius and MRSP in clinical samples than PCR. This assay has the potential to be used in small veterinary clinics due to the simplicity of the instrumental requirement. To carry out the assay, the operator only needs an isothermal device (or use body heat for 37 °C incubation) and a blue light machine, which are low-cost. In addition, this assay could be further integrated into a lateral flow strip test or a microfluidic system.

We only used RPA-CRISPR/Cas12a to directly detect the skin and ear canal samples of dogs and cats, and no samples from other sources were detected. Because RPA is tolerant to common PCR inhibitors such as 15–20% milk, 20 g/L hemoglobin, 4% (V/V) ethanol, 0.5 U heparin, serum, 1.25% urine, and a certain concentration of background DNA [35]. In the future, we can broaden the usage of this detection by using it to detect other kinds of clinical samples, such as blood, synovial fluid, urine, lung lavage fluid, etc.

However, this detection has some limitations. Firstly, RPA reagents are available from TwistDx™, but they are expensive. RPA reagents are now available in liquid form which allows for greater flexibility in reaction conditions. We still obtained good results despite reducing the 50 μL reaction system specified in the instructions to 12.5 μL which significantly lowered the detection cost. Secondly, although S. pseudintermedius is the most common bacterial pathogen isolated from clinical samples of dogs [2], they also carry other Staphylococcus such as S. schleiferi and S. aureus which can also carry the mecA gene [40]. When we directly test clinical samples with positive results for spsJ and mecA genes, the S. pseudintermedius in clinical samples could be MSSP and the mecA gene may be carried by other Staphylococcus, yielding inaccurate results. Although this has not yet occurred in any of the collected clinical samples, and there was a 100% coincidence rate between the data produced from this approach and the gold standard, the result should be interpreted with caution due to the limited clinical samples studied. In the follow-up, we should collect a large number of clinical samples to evaluate the effect of this method for direct detection of clinical samples.

Conclusion

In this study, we combined RPA and CRISPR/Cas12a to establish a rapid and accurate assay for detecting S. pseudintermedius and MRSP in clinical samples from dogs and cats. With simple instrumentation, easy-to-read results, and high sensitivity and specificity, the entire reaction can be finished within 40 min. We demonstrated the method's applicability to clinical testing with 47 clinical samples. This method can be used for diagnosing S. pseudintermedius and MRSP infections in dogs and cats to guide the rational use of antibiotics.

Materials and methods

Materials and reagents

The S. aureus BNCC337371 (methicillin-resistant) used in this study was purchased from BeNa Culture Collection (Henan, China), and all other strains involved in this study were preserved in our laboratory (National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, China) The specific information of the strains was listed in Table S1. Francisella novicida Cas12a (FnCas12a) was pre-expressed and purified in our lab as previously described [41]. TwistAmp® Liquid Basic kit was purchased from TwistDX (Cambridge, UK). HiScribe™ T7 High Yield RNA Synthesis kit, NEBuffer™ r3.1, and DNase I were provided by New England BioLabs (Beijing, China). E.Z.N.A. MicroElute RNA Clean-up kit was purchased from Omega Bio-Tek (Georgia, USA). RNase inhibitor was purchased from Takara Biomedical Technology Co. (Beijing, China). TIANgel Purification kit was purchased from Tiangen Biotech Co (Beijing, China). The synthesis of primers and ssDNA reporter, as well as the gene sequencing, were completed by Sangon Biotech (Shanghai, China). Tecan Infinite 200 PRO was purchased from Tecan Trading AG (Männedorf, Switzerland). Blue LED transilluminator was purchased from Shanghai Life Lab Biotech Co. (Shanghai, China).

Culture of bacteria and extraction of genome

The bacteria were inoculated on Columbia Blood Agar (Aobox, Beijing, China) and cultured for 24 h at 37 °C. A single colony was placed in 45 μL DNA extraction buffer (50 mM NaOH), heated at 95 °C for 5 min, and added 5 μL of 1 M Tris–HCl (pH 8.0). The mixture was centrifuged at 12,000 g for 1 min. and the supernatant was the extracted crude genomic DNA of strains. The similar method was used to extract the DNA from clinical samples. Briefly, swabs for collecting clinical samples were placed in 180 μL of DNA extraction buffer and heated at 95 °C for 5 min, then 20 μL of Tris–HCl was added and centrifuged at 12,000 g for 1 min, The supernatant was crude genomic DNA of clinical samples.

Design and synthesis of PCR primers, RPA primers, crRNAs, and positive recombinant plasmids

The spsJ gene and the methicillin resistance gene mecA gene of S. pseudintermedius (GenBank access: CP031561) were used as target genes to design PCR primers (PCR-spsJ-F/R, PCR-mecA-F/R) and RPA primers [RPA-spsJ-F/R (1, 2), RPA-mecA-F/R (1, 2)]. The specific crRNA (crRNA-spsJ1-3, crRNA-mecA1-3) primers were designed according to the amplification sequences of PCR and RPA primers (Table S3).

To prepare crRNAs, full-length crRNA primers carrying the T7 promoter were annealed and employed as RNA transcription templates. To synthesize crRNAs, transcription was performed overnight at 37 °C using T7 High Yield Transcription kit. After transcription, the DNA templates were digested with DNase I. E.Z.N.A. MicroElute RNA Clean-up kit was used to purify the crRNAs.

To create the spsJ and mecA recombinant plasmids, the spsJ and mecA genes of S. pseudintermedius were employed as the target genes. Partial sequences containing the target sequences of the spsJ and mecA genes were constructed into pUC57 backbone plasmids. Table S4 showed the partial sequences of spsJ and mecA contained in the recombinant plasmids.

PCR and RPA reaction

Each PCR reaction (25 μL) contained 12.5 μL 2 × TIANGE® Taq PCR Mix, 1 μL of each forward and reverse primers (10 μM), 1 μL of DNA template, and 9.5 μL DNase/RNase-free deionized water. The reaction conditions were as follows: predenaturation at 95 °C for 3 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 20 s. and final extension at 72 °C for 5 min. The reaction product was purified using TIANgel Purification kit.

The RPA was performed using the TwistAmp® Liquid Basic kit according to the instructions, with might modifications. Each RPA reaction (12.5 μL) contained a premix of 6.25 μL of 2 × Reaction Buffer, 0.9 µL of dNTPs (25mM), 1.25 µL of 10 × E-mix, 0.6 µL each of forward and reverse RPA primers, 0.625 µL of 20 × Core Reaction mix, 0.5 µL of DNA templates, 1.15 µL of DNase/RNase-free deionized water, and 0.625 µL of MgOAc was added at the end. The system mixture was incubated at 37 °C for 15 min to obtain the amplified products. Confirmation of the amplification product using agarose gel electrophoresis required that the RPA reaction product be heated at 65 °C for 10 min.

Establishment of RPA-CRISPR/Cas12a detection

CrRNA-spsJ1-3 and crRNA-mecA1-3 were used in CRISPR/Cas12a reactions. Each CRISPR/Cas12a reaction (20 μL) contained 30 nM Cas12a, 2 μM crRNA, 1 µL RPA amplification product, 500 nM ssDNA, 0.5 µL RNase inhibitor, 1.5 μL NEB r3.1 buffer, and DNase/RNase-free deionized water replenished the total volume to 20 μL. The system was placed in fluorescence microplate reader (Tecan Infinite 200 PRO), and the real-time fluorescence intensity was detected at 37 °C (excitation wavelength, 480 nm; emission wavelength, 520 nm). Alternatively, the reaction system was placed at 37 °C for 15 min, the reaction products were placed under blue light, the fluorescence was observed with the naked eye and photos were taken with a camera, and the endpoint fluorescence intensity of the reaction for 15 min was measured using fluorescence microplate reader.

Sensitivity of RPA-CRISPR/Cas12a detection system

The spsJ and mecA plasmids were extracted, purified and quantified by Nanodrop 2000C (Thermo Fisher Scientific, Waltham, MA, USA). The copy number was calculated, and the plasmids were diluted to a concentration of 106–100 copies per μL in a 10-fold gradient using DNase/RNase-free deionized water. The plasmids with different copy numbers were used as templates for the RPA-CRISPR/Cas12a detection.

Specificity of RPA-CRISPR/Cas12a detection system

To verify the specificity of the RPA-CRISPR/Cas12a assay for detecting spsJ and mecA genes, genomic DNA extracted from 10 MRSP strains, 10 MSSP strains, 6 Staphylococcus strains of non-S. pseudintermedius and 7 non-Staphylococcus strains using DNA extraction buffer were used for RPA-CRISPR/Cas12a reaction. The specific information of the strains was listed in Table S1.

Detection of clinical samples

We collected 47 clinical samples from China Agricultural University Veterinary Teaching Hospital (Beijing, China), including 21 samples from the skin and 26 samples from the ear canal. All clinical samples were collected with the consent of pet owners. Clinical samples were obtained by repeatedly wiping the lesion site or collecting the lesion secretions using sterile swabs. As gold standards, the sample swabs were inoculated on MSA and cultured at 37 °C for 24 h. PCR reactions were performed on yellow colonies on MSA using PCR-spsJ-F/R and PCR-mecA-F/R primers. After that, PCR products were sequenced and compared on the NCBI website to identify whether the clinical samples were cultured with MRSP or MSSP (culture + PCR). If both spsJ and mecA genes were detected in the colonies, the colonies were considered as MRSP and the corresponding clinical samples contained MRSP. If only the spsJ gene but not the mecA gene was detected, the colonies were considered MSSP, and the corresponding clinical samples contained MSSP. If no colonies were cultured or the spsJ gene was not detected from the colonies, the corresponding clinical samples did not contain S. pseudintermedius. This result was used as the gold standard. To evaluate the effect of the RPA-CRISPR/Cas12a detection system in clinical samples, sample swabs were placed in DNA extraction buffer to extract crude genomic DNA which was directly detected by RPA-CRISPR/Cas12a and direct PCR.

Statistical analysis

All experimental results were shown as mean average with standard deviation (SD) with triplicates, and data were processed by one-way ANOVA using GraphPad Prism 8.0. ***P < 0.001 was considered to indicate a statistically significant difference.

References

  1. Weese SJ, Duijkeren EV. Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in veterinary medicine. Vet Microbiol. 2010;140(3–4):418–29. https://doi.org/10.1016/j.vetmic.2009.01.039.

    Article  CAS  PubMed  Google Scholar 

  2. Bannoehr J, Guardabassi L. Staphylococcus pseudintermedius in the dog: taxonomy, diagnostics, ecology, epidemiology and pathogenicity. Vet Dermatol. 2012;23(4):253–66, e251-252. https://doi.org/10.1111/j.1365-3164.2012.01046.x.

    Article  PubMed  Google Scholar 

  3. Lynch SA, Helbig KJ. The complex diseases of Staphylococcus pseudintermedius in canines: where to next? Vet Sci. 2021;8(1):11. https://doi.org/10.3390/vetsci8010011.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Hoovels LV, Vankeerberghen A, Boel A, Vaerenbergh KV, Beenhouwer HD. First case of Staphylococcus pseudintermedius infection in a human. J Clin Microbiol. 2006;44(12):4609–12. https://doi.org/10.1128/JCM.01308-06.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Moses IB, Santos FF, Gales AC. Human colonization and infection by Staphylococcus pseudintermedius: an emerging and underestimated zoonotic pathogen. Microorganisms. 2023;11(3):581. https://doi.org/10.3390/microorganisms11030581.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cuny C, Layer-Nicolaou F, Weber R, Kock R, Witte W. Colonization of dogs and their owners with Staphylococcus aureus and Staphylococcus pseudintermedius in households, veterinary practices, and healthcare facilities. Microorganisms. 2022;10(4):677. https://doi.org/10.3390/microorganisms10040677.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lozano C, Rezusta A, Ferrer I, Perez-Laguna V, Zarazaga M, Ruiz-Ripa L, et al. Staphylococcus pseudintermedius human infection cases in Spain: dog-to-human transmission. Vector Borne Zoonotic Dis. 2017;17(4):268–70. https://doi.org/10.1089/vbz.2016.2048.

    Article  PubMed  Google Scholar 

  8. Small C, Beatty N, Helou GE. Staphylococcus pseudintermedius bacteremia in a lung transplant recipient exposed to domestic pets. Cureus. 2021;13(5):e14895. https://doi.org/10.7759/cureus.14895.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Somayaji R, Priyantha MA, Rubin JE, Church D. Human infections due to Staphylococcus pseudintermedius, an emerging zoonosis of canine origin: report of 24 cases. Diagn Microbiol Infect Dis. 2016;85(4):471–6. https://doi.org/10.1016/j.diagmicrobio.2016.05.008.

    Article  CAS  PubMed  Google Scholar 

  10. Loeffler A, Linek M, Moodley A, Guardabassi L, Sung JM, Winkler M, et al. First report of multiresistant, mecA-positive Staphylococcus intermedius in Europe: 12 cases from a veterinary dermatology referral clinic in Germany. Vet Dermatol. 2007;18(6):412–21. https://doi.org/10.1111/j.1365-3164.2007.00635.x.

    Article  PubMed  Google Scholar 

  11. Couto N, Monchique C, Belas A, Marques C, Gama LT, Pomba C. Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period. J Antimicrob Chemother. 2016;71(6):1479–87. https://doi.org/10.1093/jac/dkw029.

    Article  CAS  PubMed  Google Scholar 

  12. Nielsen SS, Bicout DJ, Calistri P, Canali E, Drewe JA, Garin-Bastuji B, et al. Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): antimicrobial-resistant Staphylococcus pseudintermedius in dogs and cats. EFSA J. 2022;20(2):e07080. https://doi.org/10.2903/j.efsa.2022.7080.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Chambers HF. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev. 1997;10(4):781–91. https://doi.org/10.1128/CMR.10.4.781.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Brown DF, Edwards DI, Hawkey PM, Morrison D, Ridgway GL, Towner KJ, et al. Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). J Antimicrob Chemother. 2005;56(6):1000–18. https://doi.org/10.1093/jac/dki372.

    Article  CAS  PubMed  Google Scholar 

  15. Adiguzel MC, Schaefer K, Rodriguez T, Ortiz J, Sahin O. Prevalence, mechanism, genetic diversity, and cross-resistance patterns of Methicillin-Resistant Staphylococcus isolated from companion animal clinical samples submitted to a veterinary diagnostic laboratory in the midwestern United States. Antibiotics (Basel). 2022;11(5):609. https://doi.org/10.3390/antibiotics11050609.

    Article  CAS  PubMed  Google Scholar 

  16. Krapf M, Muller E, Reissig A, Slickers P, Braun SD, Muller E, et al. Molecular characterisation of methicillin-resistant Staphylococcus pseudintermedius from dogs and the description of their SCCmec elements. Vet Microbiol. 2019;233:196–203. https://doi.org/10.1016/j.vetmic.2019.04.002.

    Article  CAS  PubMed  Google Scholar 

  17. Skov R, Varga A, Matuschek E, Ahman J, Bemis D, Bengtsson B, et al. EUCAST disc diffusion criteria for the detection of mecA-Mediated beta-lactam resistance in Staphylococcus pseudintermedius: oxacillin versus cefoxitin. Clin Microbiol Infect. 2020;26(1):122 e121-122 e126. https://doi.org/10.1016/j.cmi.2019.05.002.

    Article  CAS  Google Scholar 

  18. Kohner P, Uhl J, Kolbert C, Persing D, Cockerill F. Comparison of susceptibility testing methods with mecA gene analysis for determining oxacillin (methicillin) resistance in clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus spp. J Clin Microbiol. 1999;37(9):2952–61. https://doi.org/10.1128/JCM.37.9.2952-2961.1999.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sasaki T, Tsubakishita S, Tanaka Y, Sakusabe A, Ohtsuka M, Hirotaki S, et al. Multiplex-PCR method for species identification of coagulase-positive staphylococci. J Clin Microbiol. 2010;48(3):765–9. https://doi.org/10.1128/JCM.01232-09.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Verstappen KM, Huijbregts L, Spaninks M, Wagenaar JA, Fluit AC, Duim B. Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences. PLoS One. 2017;12(8):e0183925. https://doi.org/10.1371/journal.pone.0183925.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bannoehr J, Franco A, Iurescia M, Battisti A, Fitzgerald JR. Molecular diagnostic identification of Staphylococcus pseudintermedius. J Clin Microbiol. 2009;47(2):469–71. https://doi.org/10.1128/JCM.01915-08.

    Article  CAS  PubMed  Google Scholar 

  22. Nisa S, Bercker C, Midwinter AC, Bruce I, Graham CF, Venter P, et al. Combining MALDI-TOF and genomics in the study of methicillin resistant and multidrug resistant Staphylococcus pseudintermedius in New Zealand. Sci Rep. 2019;9(1):1271. https://doi.org/10.1038/s41598-018-37503-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bibby HL, Brown KL. Identification of Staphylococcus pseudintermedius isolates from wound cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry improves accuracy of susceptibility reporting at an increase in cost. J Clin Microbiol. 2021;59(11):e0097321. https://doi.org/10.1128/JCM.00973-21.

    Article  PubMed  Google Scholar 

  24. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163(3):759–71. https://doi.org/10.1016/j.cell.2015.09.038.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chen JS, Ma E, Harrington LB, Costa MD, Tian X, Palefsky JM, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science (New York, NY). 2018;360(6387):436–9. https://doi.org/10.1126/science.aar6245.

    Article  CAS  PubMed  Google Scholar 

  26. Dronina J, Samukaite-Bubniene U, Ramanavicius A. Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review). J Nanobiotechnology. 2022;20(1):41. https://doi.org/10.1186/s12951-022-01246-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mustafa MI, Makhawi AM. SHERLOCK and DETECTR: CRISPR-Cas Systems as potential rapid diagnostic tools for emerging infectious diseases. Am Soc Microbiol. 2021;59(3):e00745-00720. https://doi.org/10.1128/JCM.

    Article  CAS  Google Scholar 

  28. Li SY, Cheng QX, Wang JM, Li XY, Zhang ZL, Gao S, et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4(20). https://doi.org/10.1038/s41421-018-0028-z

  29. Woźniakowski G, Fu J, Zhang Y, Cai G, Meng G, Shi S. Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus. Plos One. 2021;16(7):e0254815. https://doi.org/10.1371/journal.pone.0254815.

    Article  CAS  Google Scholar 

  30. Mahas A, Hassan N, Aman R, Marsic T, Wang Q, Ali Z, et al. LAMP-Coupled CRISPR-Cas12a module for rapid and sensitive detection of plant DNA viruses. Viruses. 2021;13(3):466. https://doi.org/10.3390/v13030466.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang Y, Li J, Li S, Zhu X, Wang X, Huang J, et al. LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test. Mikrochim Acta. 2021;188(10):347. https://doi.org/10.1007/s00604-021-04985-w.

    Article  CAS  PubMed  Google Scholar 

  32. Shi Y, Kang L, Mu R, Xu M, Duan X, Li Y, et al. CRISPR/Cas12a-enhanced loop-mediated isothermal amplification for the visual detection of Shigella flexneri. Front Bioeng Biotechnol. 2022;21(10):845688. https://doi.org/10.3389/fbioe.2022.845688.

    Article  Google Scholar 

  33. Phumthanakorn N, Chanchaithong P, Prapasarakul N. Development of a set of multiplex PCRs for detection of genes encoding cell wall-associated proteins in Staphylococcus pseudintermedius isolates from dogs, humans and the environment. J Microbiol Methods. 2017;142:90–5. https://doi.org/10.1016/j.mimet.2017.09.003.

    Article  CAS  PubMed  Google Scholar 

  34. Morris DO, Loeffler A, Davis MF, Guardabassi L, Weese JS. Recommendations for approaches to meticillin-resistant staphylococcal infections of small animals: diagnosis, therapeutic considerations and preventative measures. Clinical Consensus Guidelines of the World Association for Veterinary Dermatology. Vet Dermatol. 2017;28(3):304-e369. https://doi.org/10.1111/vde.12444.

    Article  PubMed  Google Scholar 

  35. Daher RK, Stewart G, Boissinot M, Bergeron MG. Recombinase polymerase amplification for diagnostic applications. Clin Chem. 2016;62(7):947–58. https://doi.org/10.1373/clinchem.2015.245829.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Daher RK, Stewart G, Boissinot M, Boudreau DK, Bergeron MG. Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology. Mol Cell Probes. 2015;29(2):116–21. https://doi.org/10.1016/j.mcp.2014.11.005.

    Article  CAS  PubMed  Google Scholar 

  37. Higgins M, Stringer OW, Ward D, Andrews JM, Forrest MS, Campino S, et al. Characterizing the Impact of primer-template mismatches on recombinase polymerase amplification. J Mol Diagn. 2022;24(11):1207–16. https://doi.org/10.1016/j.jmoldx.2022.08.005.

    Article  CAS  PubMed  Google Scholar 

  38. Boyle DS, Lehman DA, Lillis L, Peterson D, Singhal M, Armes N, et al. Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification. mBio. 2013;4(2):10–128. https://doi.org/10.1128/mBio.00135-13.

    Article  Google Scholar 

  39. Ling C, Chang Y, Wang X, Cao X, Tu Q, Liu B, et al. Two CRISPR/Cas12a-based methods for fast and accurate detection of single-base mutations. Anal Chim Acta. 2023;1247:340881. https://doi.org/10.1016/j.aca.2023.340881.

    Article  CAS  PubMed  Google Scholar 

  40. Lakhundi S, Zhang K. Methicillin-Resistant Staphylococcus aureus: Molecular characterization, evolution, and epidemiology. Clin Microbiol Rev. 2018;31(4):e00020-18. https://doi.org/10.1128/CMR.00020-18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wang L, Fu J, Cai G, Cheng X, Zhang D, Shi S, et al. Rapid and visual RPA-Cas12a fluorescence assay for accurate detection of dermatophytes in cats and dogs. Biosensors (Basel). 2022;12(8):636. https://doi.org/10.3390/bios12080636.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Jinyu Fu (Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China) for her help to our work. We also thank China Agricultural University Veterinary Teaching Hospital for providing the clinical samples of dogs and cats. We are grateful to the pet owners for their cooperation in sampling.

Funding

This research was funded by the National Natural Science Foundation of China (32100156), and China Agricultural University Teaching Animal Hospital Talent Funding.

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Authors and Affiliations

  1. National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China

    Pingping Gao, Di Zhang & Yueping Zhang

  2. Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China

    Shuobo Shi

  3. Beijing Zhongnongda Veterinary Hospital Co., Ltd, Beijing, 100193, China

    Di Zhang

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  1. Pingping Gao
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Contributions

P.G., D.Z., and Y.Z. conceived and designed the experiments; S.S. provided the methodology and discussion. P.G. performed the experiments and analyzed the data; P.G. and Y.Z. wrote the manuscript; Y.Z. and D.Z. participated in critical review of the manuscript. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Di Zhang or Yueping Zhang.

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Ethics approval and consent to participate

The study protocol was approved by the China Agricultural University Laboratory Animal Welfare and Animal Experimental Ethical Committee (Issue No. AW11503202-2–5).

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There is no conflict of financial interests.

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Supplementary Information

Additional file 1: Fig. S1.

Endpoint fluorescence intensities of RPA products involved in Cas12a reactions at different reaction times. Fig. S2. Results for direct detection of the spsJ and mecA genes in 47 clinical samples by direct PCR. Table S1. Bacterial strains used in specificity of RPA-CRISPR/Cas12a detection. Table S2. Gold standard, RPA-CRISPR/Cas12a and direct PCR results of 47 clinical samples. Table S3. The primer sequences used in this study. Table S4. The spsJ and mecA gene sequences contained in the recombinant plasmids.

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Gao, P., Shi, S., Zhang, D. et al. Rapid and visual RPA-CRISPR/Cas12a detection for Staphylococcus pseudintermedius and methicillin-resistant S. pseudintermedius in clinical samples of dogs and cats. One Health Adv. 1, 20 (2023). https://doi.org/10.1186/s44280-023-00021-z

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One Health Advances

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