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

ABSTRACT The monkeypox virus, which was declared as a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO), continues to cause infection cases worldwide. Given the risk of virus evolution, it is essential to identify monkeypox virus infection in a timely manner and isolate patients to prevent outbreaks so that the vulnerable population is protected and the emergence of dangerous monkeypox variants is restrained. In this study, a novel one-pot recombinase polymerase amplification-Clustered Regularly Interspaced Short Palindromic Repeats (RPA-CRISPR) assay for monkeypox based on a sucrose-aided multiphase aqueous system has been established with an ultra-high sensitivity of a single copy, which is 10 times higher than the existing RPA-CRISPR one-pot method. The entire reaction was completed within 60 min at 37°C. The detection method exhibited good specificity, effectively distinguishing monkeypox from other orthopoxviruses. The detection results could be observed by the naked eye under ultraviolet or blue light, making it highly suitable for home or limited healthcare settings. The assay has solved the incompatibility between the Cas12a cleavage reaction and the RPA reaction and shows good specificity, accuracy, and the rapidness and convenience important for point-of-care testing. It provides an effective tool for the early diagnosis of monkeypox and sets a technical example of using a multiphase aqueous system to establish one-pot RPA-CRISPR detection. IMPORTANCE The monkeypox virus was declared as a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO) and continues to cause infection cases worldwide. Given the risk of virus evolution, it is essential to identify monkeypox virus infection in a timely manner to prevent outbreaks. This study establishes a novel one-pot recombinase polymerase amplification-Clustered Regularly Interspaced Short Palindromic Repeats (RPA-CRISPR) assay for monkeypox virus with an ultra-high sensitivity. The assay shows good specificity, accuracy, and the rapidness and convenience important for point-of-care testing. It provides an effective tool for the early diagnosis of monkeypox, which is useful for the prevention of an epidemic.

The manuscript proposes a new method for the monkeypox virus detection.Due to the risk of monkeypox virus transmission and evolution, timely detection of monkeypox virus infection is essential to prevent monkeypox virus outbreaks.The authors developed a one-pot RPA-CRISPR assay for monkeypox based on a sucrose-aided multiphase aqueous system has been established with an ultra-high sensitivity of single copy, which is 10 times higher than the existing RPA-CRISPR one-pot method.The approach for monkeypox virus detection is valuable.However, there are still some issues should be addressed.1.The manuscript established a sucrose-aided multiphase aqueous system for the one-pot detection of the monkeypox virus.By taking advantage of the density difference between sucrose and water, the separation of the RPA reaction and the CRISPR reaction within the same tube was realized.Can other reagents be used instead of sucrose?In addition, please discuss why sucrose does not interfere with protease response.2. The authors optimized the concentrations of sucrose and the proportion of the RPA reaction system and CRISPR reaction system.But the concentrations of gRNA and Cas12a are important for CRISPR/system.Please explain the concentrations and basis of gRNA and Cas protein use.3. The manuscript compared the methods of recent studies on molecular detection of monkeypox, but some other Cas12abased method should be discussed.For example, ACS Sens. 2021, 6, 9, 3295-3302;Nat. Biotechnol. 2020, 38, 870-874;J. Agric. Food Chem. 2021, 69, 35, 10321-10328;Nano Lett. 2021, 21, 19, 8393-8400. 4. The P values from significance analysis is suggested to provide in sensitivity test. 5.The expression and purification of Cas12a protein should be descripted with more details.6.More information about the selection of target RNA sequence is suggested to be provided.7. If possible, please test the clinical samples using the method.
Reviewer #2 (Comments for the Author): 1.The monkeypox is no longer a Public Health Emergency of International Concern as announced by WHO in May 2023.But it continues to pose a major public health challenge that requires a strong, proactive, and sustainable response.The authors have to update the information about the monkeypox epidemic and restate the significance of this study relating to the current situation.
2. There are several molecular detection methods using CRISPR in the literature.Also, there are RPA-based assays reported with high sensitivity.This study claimed an improvement on sensitivity with the one-pot CRISPR assay.The benefits of the higher sensitivity, the one-pot assay, and the use of CRISPR enzyme have to be further discussed to show the application advantage of this study.
3. The method established in this study is claimed to be used for POCT.However, DNA extraction has to be performed, and the authors described using a qPCR machine to read the signals.How would these be conducted in POCT?The authors have to clarify how to set up this assay in POCT, including the equipment and necessary training required.
4. The simulated samples used the plasmid with the monkeypox target sequence.This is an incomplete simulation because virus lysis and extraction of the genome are not covered.It is preferred to further validate the method with at least the pseudovirus.
5. Some of the experimental procedures and figures are not described with enough clarity.Suggest to improve the language of the Methods and Figure legends.6.In Figure 6 A and B, the signal intensity of the sample with 10^2 copies is not consistent in the curve and the bar, please check the accuracy of the data.

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Response to Reviewers: (responses in blue)
Reviewer #1 (Comments for the Author): The manuscript proposes a new method for the monkeypox virus detection.Due to the risk of monkeypox virus transmission and evolution, timely detection of monkeypox virus infection is essential to prevent monkeypox virus outbreaks.The authors developed a one-pot RPA-CRISPR assay for monkeypox based on a sucrose-aided multiphase aqueous system has been established with an ultra-high sensitivity of single copy, which is 10 times higher than the existing RPA-CRISPR one-pot method.The approach for monkeypox virus detection is valuable.However, there are still some issues should be addressed.We thank the positive comments from the reviewer.
1.The manuscript established a sucrose-aided multiphase aqueous system for the one-pot detection of the monkeypox virus.By taking advantage of the density difference between sucrose and water, the separation of the RPA reaction and the CRISPR reaction within the same tube was realized.Can other reagents be used instead of sucrose?In addition, please discuss why sucrose does not interfere with protease response.Glycerol can be used instead of sucrose.Sucrose and glycerol are traditional enzyme protectors, and studies have shown their enhancing effect on nucleic acid detections.This discussion has been added to the last paragraph of section "Principle of sucrose-aided multiphase one-pot RPA-CRISPR reaction" of RESULTS AND DISCUSSION.
2. The authors optimized the concentrations of sucrose and the proportion of the RPA reaction system and CRISPR reaction system.But the concentrations of gRNA and Cas12a are important for CRISPR/system.Please explain the concentrations and basis of gRNA and Cas protein use.Regarding to the concentrations of Cas12a and crRNA, the literatures and the instruction of commercialized Cas12a (New England Biolabs) have recommended a molar ratio of ~1:1 in the range of 30-50 nM final.This study selected the molar ratio of 1:1 at 33 nM final for Cas12a and crRNA concentrations.This discussion has been added to the last paragraph of section "Construction of the one-pot RPA-CRISPR reaction in a sucrose-aided multiphase aqueous system" of RESULTS AND DISCUSSION.
4. The P values from significance analysis is suggested to provide in sensitivity test.The P values have been added to Figure 6B of the revised manuscript.
5. The expression and purification of Cas12a protein should be descripted with more details.The result of expression and purification of Cas12a is shown in Figure S2.More details have been added to section "Expression and purification of Cas12a" of MATERIALS AND METHODS.
6.More information about the selection of target RNA sequence is suggested to be provided.The detailed information about the selection of PAM motifs and the design of crRNAs are provided in Figure S1, and described with better clarity in section "crRNA preparation" of MATERIALS AND METHODS of the revised manuscript.
7. If possible, please test the clinical samples using the method.Because of the strict control of the monkeypox virus clinical samples, we chose to use the monkeypox pseudovirus and validated the method with simulated samples.The results are presented in Figure 7 of the revised manuscript.

Reviewer #2 (Comments for the Author):
1.The monkeypox is no longer a Public Health Emergency of International Concern as announced by WHO in May 2023.But it continues to pose a major public health challenge that requires a strong, proactive, and sustainable response.The authors have to update the information about the monkeypox epidemic and restate the significance of this study relating to the current situation.Changes have been made to the revised manuscript with the updated information in ABSTRACT, IMPORTANCE and the first paragraph of INTRODUCTION.
2. There are several molecular detection methods using CRISPR in the literature.Also, there are RPA-based assays reported with high sensitivity.This study claimed an improvement on sensitivity with the one-pot CRISPR assay.The benefits of the higher sensitivity, the one-pot assay, and the use of CRISPR enzyme have to be further discussed to show the application advantage of this study.Application of this one-pot RPA-CRISPR method has three advantages: (1) the high sensitivity reduces false-negative results and helps early diagnosis of an infection; (2) the amplification-cleavage system reduces false-positive results and gives better diagnosis accuracy; (3) the one-pot setting avoids exposing the amplified materials to the environment and reduces cross-contaminations.A discussion has been added to the second last paragraph of section "Advances on technology and application" of RESULTS AND DISCUSSION of the revised manuscript.
3. The method established in this study is claimed to be used for POCT.However, DNA extraction has to be performed, and the authors described using a qPCR machine to read the signals.How would these be conducted in POCT?The authors have to clarify how to set up this assay in POCT, including the equipment and necessary training required.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 or a portable fluorescence reader.These supplies are easy to assemble, making the method highly suitable for POCT.A discussion has been added to the second last paragraph of section "Advances on technology and application" of RESULTS AND DISCUSSION of the revised manuscript.
4. The simulated samples used the plasmid with the monkeypox target sequence.This is an incomplete simulation because virus lysis and extraction of the genome are not covered.It is preferred to further validate the method with at least the pseudovirus.Because of the strict control of the monkeypox virus clinical samples, we chose to use the monkeypox pseudovirus and validated the method with simulated samples.The results are presented in Figure 7 of the revised manuscript.
5. Some of the experimental procedures and figures are not described with enough clarity.Suggest to improve the language of the Methods and Figure legends.The language of the Methods and Figure legends has been modified in the revised manuscript.We believe the language is now satisfactory, presenting information with good clarity.6.In Figure 6 A and B, the signal intensity of the sample with 10^2 copies is not consistent in the curve and the bar, please check the accuracy of the data.We had made a mistake in the bar chat of Figure 6B.It is now corrected in the revised manuscript.
November 12, 2023 1st Revision -Editorial Decision Re: Spectrum02267-23R1 (Ultrasensitive one-pot detection of monkeypox virus with RPA and CRISPR in a sucrose-aided multiphase aqueous system) Dear Dr. Song Gao: The Reviewers' comments have been adequately addressed.
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Sincerely, Frederick S. Kibenge Editor Microbiology Spectrum Reviewer #1 (Comments for the Author): Authors have addressed the issues.