Ultrasensitive one-pot detection of monkeypox virus with RPA and CRISPR in a sucrose-aided multiphase aqueous system

The combination of RPA and CRISPR technology in the development of a one-pot detection method often leads to a loss of sensitivity, which may be due to the incompatibility between Cas12a cleavage and RPA (25, 26). In the early stage of the one-pot reaction, Cas12a can form a complex with specific targets under the guidance of crRNA, thereby activating the cis and trans cleavage activities of Cas12a. The specific cis cleavage activity leads to the loss of RPA template DNA and the difficulty in accumulating newly generated RPA amplicons. The non-specific trans cleavage activity leads to the cleavage of primers in the reaction system, resulting in reduced RPA efficiency. For example, Mao et al. reported a two-step method for detecting monkeypox using RPA combined with Cas12a, with a sensitivity of 10⁰ copies per reaction (23). In contrast, Chen et al. established a one-step method for detecting monkeypox using RPA combined with Cas12a, with a sensitivity of 15 copies per reaction (24).

In this study, to address the challenge of overcoming the incompatibility between the RPA reaction and the CRISPR reaction, we established a sucrose-aided multiphase aqueous system for the one-pot detection of the monkeypox virus (Fig. 1). By taking advantage of the density difference between sucrose and water, we were able to spatially separate the RPA reaction and the CRISPR reaction within the same tube. The RPA reaction, with the addition of sucrose to increase its density, was placed at the bottom of the tube, while the CRISPR reaction, with lower density, remained in the water phase and was positioned on top of the RPA reaction. During the one-pot reaction, slow and dynamic spreading occurred between the two phases. The newly accumulated RPA amplicons triggered the activation of the non-specific trans cleavage of Cas12a, resulting in the cleavage of ssDNA-FQ and the release of the fluorophore; meanwhile, the phase separation diminished the effect on RPA. Real-time fluorescence signals were detected using a fluorescence detector, while the end-point fluorescence could be visualized under a blue light source.

Various methods based on the principle of multiphase aqueous systems have been reported to overcome the compatibility issue between RPA and CRISPR cleavage (27, 28). Yin et al. reported a molecular diagnostic method for CRISPR-Cas12a using a multiphase aqueous system, dynamic aqueous multiphase reaction (DAMR), which utilizes the density difference based on sucrose concentration (28). In the DAMR system, RPA- and CRISPR-Cas12a-derived fluorescence detections occur in spatially separated but connected aqueous phases. This method detected 10 copies of HPV16 and 100 copies of HPV18 in less than 1 h (28). Lin et al. reported a one-pot RPA-CRISPR method developed using a multiphase aqueous system based on the density difference associated with glycerol concentration. This method demonstrated a 100-fold improvement in sensitivity compared to the glycerol-free approach (27). These examples demonstrated the applicability of density-based multiphase systems for solving the RPA-CRISPR compatibility issue. Sucrose and glycerol are traditional enzyme protectors, and studies have shown their enhancing effect on nucleic acid detection (29, 30). Our study further proved that sucrose would not interfere with CRISPR cleavage.

The three forward primers (F1–F3) and three reverse primers (R1–R3) (Table 1) could make nine primer pair combinations, which were tested in PCRs. The results of the 1.5% agarose gel showed that the amplification efficiency of the reactions using the combinations of F1R1, F1R2, and F3R2 was higher than that of the other combinations (Fig. 2A). Subsequently, the three primer pairs were individually re-confirmed in the RPA reactions, and the results of the 1.5% agarose gel showed that the amplification efficiency of the RPA reaction with the primer pair F1R2 was the highest (Fig. 2B). Finally, the sensitivity of the RPA reaction with the primer pair F1R2 was tested, and the results showed that the amplification efficiency was good, with as low as 10¹ copies of the template (Fig. 2C). Therefore, the primer pair F1R2 was selected for the subsequent experiments.

After determining the RPA primer, the DNA sequence of the RPA amplicon was analyzed, and four crRNAs were designed (Table 1). To establish a highly efficient CRISPR reaction, the four crRNAs were tested individually. The results of the fluorescence curves showed that with the guidance of crRNA1, the cleavage activity of Cas12a was the highest, and the end-point fluorescence could be visualized under a blue light source (Fig. 3A). Then, the sensitivity of the two-step RPA-CRISPR method was determined by using different concentrations of the standard plasmid. Both the results of the fluorescence curves and the end-point fluorescence showed that as few as 10¹ copies of the standard plasmid could be detected (Fig. 3B).

Based on the two-step RPA-CRISPR reaction, efforts were made to establish a one-pot RPA-CRISPR reaction. First, 10⁵ copies of the standard plasmid were used as the templates and tested in the one-pot RPA-CRISPR reaction that directly combined the RPA reaction and the CRISPR reaction. However, the results showed no obvious difference between the fluorescence curves of the positive reaction and the no-template control (Fig. 4A). As we have speculated, the reason could be that in the one-pot mixture containing the RPA reagents and the CRISPR reagents, the newly produced RPA amplicons would be cleaved by Cas12a, resulting in poor accumulation of the amplicons and a low fluorescence signal (25, 26).

To solve this problem, sucrose was introduced to construct the multiphase aqueous system. The sucrose density is 1.8 ± 0.1 g/cm³, which is higher than that of water. The density of the RPA reaction with sucrose was higher than that of the CRISPR reaction in the aqueous phase. Additionally, sucrose demonstrated stability in both physical and chemical properties. Here, the sucrose-aided multiphase aqueous system was constructed with the CRISPR reaction in the upper phase and the RPA reaction in the bottom phase and was confirmed by using a template of 10⁴ copies of a standard plasmid (Fig. 4B). Indeed, the good fluorescence signal was related to the better RPA in the multiphase aqueous system. The multiphase reaction (+ sucrose) that showed a good fluorescence signal did have much better accumulation of the RPA amplicons as compared to the monophase reaction (- sucrose) (Fig. 4C and D).

After the construction of the multiphase aqueous system, a series of optimization experiments was conducted. These experiments included optimizing the concentrations of sucrose and the ratio of the RPA reaction to the CRISPR reaction. The optimization of sucrose concentrations was tested by adding different concentrations of sucrose into the RPA reactions. The results showed that the one-pot reaction with 5% sucrose in the RPA (sucrose) phase exhibited the highest fluorescence signals (Fig. 5A). For the optimization of the ratio of the RPA reaction to the CRISPR reaction, three different ratios were tested: 1.5:1, 2:1, and 3:1. The results indicated that the reaction with a ratio of 2:1 for the RPA reaction to the CRISPR reaction displayed the highest fluorescence signals (Fig. 5B). With regard to the concentrations of Cas12a and crRNA, the literature and the instructions for commercialized Cas12a (New England Biolabs) have recommended a molar ratio of ~1:1 in the range of 30–50 nM final (20, 24, 31). This study selected the molar ratio of 1:1 at 33 nM final for Cas12a and crRNA concentrations.

To evaluate the limit of detection of the sucrose-aided multiphase one-pot RPA-CRISPR method, a series of different copies of the standard plasmid of the monkeypox virus, including 10⁵, 10⁴, 10², 10¹, and 10⁰, were tested. The results of the fluorescence curves and the end-point fluorescence showed that as few as 10⁰ copies of the standard plasmid could be detected (Fig. 6A and B). The significance analysis further indicated that the positive signals were solid.

The specificity of the sucrose-aided one-pot RPA-CRISPR method was confirmed by testing a series of commonly encountered vaccinia viruses and coronaviruses, including cowpox virus, buffalopox virus, vaccinia virus, variola virus, SARS-CoV-2 N gene, SARS-CoV-2 ORF gene, and Pseudomonas aeruginosa. Both the results of the fluorescence curves and the end-point fluorescence demonstrated that this method was specific to the monkeypox virus (Fig. 6C and D).

Since the monkeypox virus is presumed to spread through close contact with bodily fluids, we prepared three simulated samples by mixing the bodily fluids from blood samples, skin swabs, and throat swabs with the monkeypox standard plasmid and one simulated sample by mixing the skin swab with the monkeypox pseudovirus. These samples were then tested to validate the sucrose-aided one-pot RPA-CRISPR method. The fluorescence curves and the end-point fluorescence results indicated that all four types of simulated samples could be detected with single-copy sensitivity, demonstrating the potential of the one-pot method for clinical diagnosis of the monkeypox virus (Fig. 7).

CRISPR-Cas12a has shown good performance in nucleic acid detection when combined with nucleic acid amplification technologies (32 – 35). The specific activation of Cas12a cleavage guided by crRNA and the signal amplification effect are the main improving factors. To make this amplification-cleavage detection system more convenient and to reduce the risk of carry-over contamination, one-pot assays are the technology development direction. Specifically, the development of RPA-CRISPR one-pot detection methods requires overcoming the compatibility issue between the RPA reaction and the CRISPR cleavage reaction. Lin et al.’s study implemented a single-tube system for the RPA and CRISPR reactions in a glycerol-based double aqueous system, improving the reaction sensitivity by 100 times (27). Yin et al. developed a double aqueous system utilizing sucrose density to overcome the incompatibility between the RPA and CRISPR reactions, enabling highly sensitive and quantitative detection (28). Gong et al. used sulfur-modified RPA primers to avoid detection failures caused by non-specific cleavage of RPA primers by Cas12a (36). Lu et al. established a rapid and highly sensitive one-pot method for the RPA and CRISPR reactions based on suboptimal protospacer adjacent motif (PAM) sequences (37). Chen et al. reported a method that temporarily inactivates crRNA by introducing photocleavable nucleotides complementary to crRNA (25). These attempts to combine the RPA and CRISPR reactions in a one-pot method provide successful cases for the application of CRISPR technology in on-site detection scenarios. In this study, we constructed the multiphase aqueous system using sucrose, enabling the stratification of the RPA reaction system and the CRISPR reaction system in a single tube with a gradual diffusion property. Ultimately, the sensitivity of the RPA-CRISPR one-pot method for monkeypox detection reached a single copy per reaction. The optimization process of the sucrose system in this study indicates a wide redundancy in concentration and ratio, making it an easy-to-use principle for one-pot RPA-CRISPR methods.

With the multiphase aqueous system, the one-pot RPA-CRISPR method for the detection of monkeypox virus showed a sensitivity of a single copy per reaction, which was higher than the existing RPA-CRISPR one-pot method by one magnitude (Table S1) (24). The detection method exhibited good specificity, effectively distinguishing monkeypox from other orthopoxviruses. Application of this one-pot RPA-CRISPR method has three advantages: (i) the high sensitivity reduces false-negative results and helps the early diagnosis of an infection; (ii) the amplification-cleavage system reduces false-positive results and gives better diagnostic accuracy; and (iii) the one-pot setting avoids exposing the amplified materials to the environment and reduces cross-contamination (19, 27, 28, 36). From the POCT perspective, the entire reaction was completed within 60 min at 37°C, and the detection results could be observed by the naked eye under ultraviolet or blue light. Although we used a qPCR machine to quantify the fluorescence signals in the method development, a portable fluorescence reader can do the quantification in real applications. For viral DNA extraction, a battery-powered mini-centrifuge is well enough. Thus, the method requires a set of pipettes, a mini-centrifuge, a low-power heat block, and a blue light source or a portable fluorescence reader. These supplies are easy to assemble, making the method highly suitable for home or limited healthcare settings.

In conclusion, this study utilized sucrose to increase the density of the RPA reaction and constructed a one-pot RPA-CRISPR method with a multiphase aqueous system, achieving rapid, convenient, and reliable detection of monkeypox virus. It assists in the prevention and control of monkeypox outbreaks and provides a good example of using a multiphase aqueous system to implement RPA-CRISPR one-pot methods.

The standard plasmid representing a portion of the monkeypox virus F3L gene (GenBank no. ON568298/ON563414, 46,168–46,630 nt) in the background of pUC57 was purchased from Sangon Biotech (Shanghai) Co. Ltd., China. Two plasmids containing the SARS-CoV-2 N gene and ORF gene and the genomic DNA of Pseudomonas aeruginosa (P. aeruginosa, ATCC 9027) were preserved in the laboratory (38). Regions of the F3L homologous genes from other human-related orthopoxviruses, including the vaccinia virus (GenBank no. S64006.1, 170–630 nt of the E3L gene), the cowpox virus (GenBank no. NC_003663.2, 64,468–64,926 nt), the variola virus (GenBank no. DQ437592.1, 39,014–39,472 nt), and the buffalopox virus (GenBank no. MG599038.1, 51,078–51,536 nt), were synthesized in the background of pET28b(+) (General Biology Co. Ltd., Anhui, China). The sequence alignment of the homologous regions is shown in Fig. S1.

The highly conserved F3L gene was selected as the detection target as described in the previous reports (39, 40). RPA primers were designed following the instructions of the RAA Nucleic Acid Amplification Reagent (Hangzhou ZC Bio-Sci & Tech Co. Ltd., Zhejiang, China) (Table 1; Fig. S1).

RPA primers were initially screened by PCR. The PCR mixture (20 µL) contained 10 µL of the 2× Taq Master Mix (Vazyme Biotech Co. Ltd., Nanjing, China), 1 µL each of the forward and reverse primers (10 µM), and 5 µL of the template. The cycle setting was pre-denaturation at 95°C for 3 min followed by 30 cycles of 15 sec at 95°C, 15 sec at 55°C, and 60 sec at 72°C. The PCR amplicons were analyzed by agarose gel electrophoresis.

The RPA reactions were conducted following the instructions of the RAA Nucleic Acid Amplification Reagent (Hangzhou ZC Bio-Sci & Tech Co. Ltd.). To a lyophilized enzyme pellet, 25 µL of A buffer, 4 µL each of the forward and reverse primers (10 µM), and 8.5 µL of deionized water were added. The mixture was split into two halves for two reactions. Into each half of the mixture, 3 µL of the template DNA was added, and 1.25 µL of B buffer was added to start the reaction. The reaction (25 µL) was incubated at 37°C for 30 min. The amplicons were analyzed by agarose gel electrophoresis.

The coding sequence of Cas12a (LbCas12a) was obtained from a standard plasmid (Addgene plasmid no. 90096). The sequence was inserted into the multiple cloning site of the pET28b(+) vector by molecular cloning techniques, resulting in a recombinant open reading frame of an N-terminal His-tagged Cas12a for overexpression. The expression was induced with 0.5 mM IPTG for 8 h at 23°C in the Escherichia coli BL21(DE3)pLysS strain. The His-tagged recombinant Cas12a was purified by Ni affinity chromatography on a Ni-NTA 6FF column (Sangon Biotech Co. Ltd.) with a linear gradient of imidazole (0–500 mM) on an AKTA Prime Plus system (GE Healthcare Life Sciences, Boston, MA, USA). The purified Cas12a was analyzed by SDS-PAGE (Fig. S2) and stored at −20°C in 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM TCEP, and 50% (vol/vol) glycerol.

The crRNAs were designed within the RPA amplicon sequences of the monkeypox virus F3L gene based on the available PAM motifs (TTTN) (Table 1; Fig. S1). For crRNA preparation, two reverse-complement ssDNA fragments that could form a dsDNA fragment containing the T7 promoter, the sequence for the crRNA skeleton, and the 21-bp sequence downstream of the corresponding PAM motif upon annealing were synthesized (General Biology Co. Ltd.). The annealed dsDNA served as the template for in vitro transcription with the T7 High Yield RNA Transcription Kit (Vazyme Biotech Co. Ltd.). The transcribed crRNA was purified by phenol-chloroform extraction, dissolved in 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC) water, and quantified by a Qubit 4 fluorometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA).

The 20-µL Cas12a cleavage reaction mixture contained the desired amount of the DNA sample and 50 nM Cas12a, 50 nM crRNA, and 500 nM ssDNA-FQ (the fluorescence reporter with FAM and BHQ1 labels at the two ends) (Table 1) in 1× NEB Buffer 2.1. For real-time fluorescence detection, the reaction was conducted at 37°C for 15 min on the Roche LightCycler 480 II qPCR machine (Basel, Switzerland) with FAM signal reading at 1-min intervals. For end-point fluorescence detection, the signal was visualized under a blue light source after incubation at 37°C for 15 min. The images were taken by a smartphone camera.

The RPA premix and the CRISPR premix were prepared separately. The RPA premix was prepared by adding 4 µL each of the forward and reverse primers (10 µM) and 18 µL of A buffer to one lyophilized enzyme pellet (Hangzhou ZC Bio-Sci & Tech Co. Ltd.). The CRISPR premix was prepared by mixing 200 nM Cas12a, 2 µM ssDNA-FQ, and 200 nM crRNA in 1× NEB Buffer 2.1. The 25-µL RPA-CRISPR one-pot reaction was prepared by mixing 14.5 µL of the RPA premix, 6.25 µL of the CRISPR premix, and 3 µL of the template DNA and initiated by the addition of 1.25 µL of B buffer. The reaction was conducted at 37°C for 60 min on the Roche LightCycler 480 II qPCR machine (Basel, Switzerland) with FAM signal reading at 1-min intervals. The end-point signal was visualized under a blue light source, and the images were taken by a smartphone camera.

The RPA premix and the CRISPR premix were prepared separately. For the RPA premix, 4 µL each of the forward and reverse primers (10 µM), 18 µL of A buffer, and 3.25 µL of sucrose (50%, vol/vol) were added to one lyophilized enzyme pellet (Hangzhou ZC Bio-Sci & Tech Co. Ltd.). The CRISPR premix was prepared by mixing 100 nM Cas12a, 1 µM ssDNA-FQ, and 100 nM crRNA in 1× NEB Buffer 2.1. To conduct the RPA-CRISPR one-pot reaction with the sucrose-aided multiphase, 18 µL of the RPA premix, 1 µL of the template, and 1 µL of B buffer were added to the bottom of the reaction tube and gently mixed. Then, 10 µL of the CRISPR premix was slowly added to the top of the above mixture, creating the sucrose-aided multiphase aqueous reaction system. The reaction was conducted at 37°C for 60 min on the Roche LightCycler 480 II qPCR machine (Basel, Switzerland) with FAM signal reading at 1-min intervals. The end-point signal was visualized under a blue light source, and the images were taken by a smartphone camera.

The skin swabs, throat swabs, and peripheral blood samples were collected from the consent-informed healthy persons. To prepare simulated skin swab and throat swab samples, the skin swabs or throat swabs were immersed in 2 mL of virus storage solution (Beyotime Biotechnology Co. Ltd., Shanghai, China), and then the solution was separated in 200-µL aliquots and mixed with the desired amounts of the monkeypox standard plasmid or the monkeypox pseudovirus (Fubio Biological Technology Co. Ltd., Shanghai, China). To prepare simulated blood samples, the collected blood samples were separated in 200-µL aliquots and mixed with desired amounts of the monkeypox standard plasmid or the monkeypox pseudovirus. DNA was extracted with the TIANamp Virus DNA/RNA Kit (Tiangen Biotech Co. Ltd., Beijing, China), and 1 µL was used for detection.