Rapid Detection of Phytophthora cambivora Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a
Round 1
Reviewer 1 Report
The manuscript by Zhou et al. presents the RPA-CRISPR/Cas12a assay for Phytophthora cambivora detection.
Regarding the novelty of the study, it appears that the figures and table in the manuscript closely resemble those published by Gou et al., 2023, especially Figure 1 and Table 1. While I understand that these works may originate from the same research group, it is essential to avoid directly duplicating.
The manuscript contains typographical, language, and grammatical issues that require attention, for example, in lines 79-80, line 122, line 211, 240, Line 243, lines 247-249, and line 303. To enhance readability and overall quality, I recommend that the authors send the manuscript for language editing before resubmitting it
Author Response
1. Regarding the novelty of the study, it appears that the figures and table in the manuscript closely resemble those published by Gou et al., 2023, especially Figure 1 and Table 1. While I understand that these works may originate from the same research group, it is essential to avoid directly duplicating.
Response 1: Thanks for your positive comments and constructive suggestions. Because we are in the same research group, the detection strains used was all shared by the research group, and our detection objects are Phytophthora, so the content of table1 will be similar. The existing duplicate strains used for detection in the table have been deleted, and the content and clarity of other figures have been adjusted.
2. The manuscript contains typographical, language, and grammatical issues that require attention, for example, in lines 79-80, line 122, line 211, 240, Line 243, lines 247-249, and line 303. To enhance readability and overall quality, I recommend that the authors send the manuscript for language editing before resubmitting it.
Response 1: Thanks for your positive comments and constructive suggestions. The grammatical errors in the text have now been corrected accordingly, as described in the original article.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript reported the popular RPA-CRISPR / Cas12a method for pathogenic diagnosis used for P. cambivora by designing primers based on the specific target gene g2339. Most of the methods are similar with the paper published by the authers(Front. Cell. Infect. Microbiol. 13:1218105.and Front. Cell. Infect. Microbiol. 13:1208837).
(1) The major problem was the author conclude that a new detection method based on RPA- CRISPR / Cas12a was established to detect P. cambivora, actually, it was not a new method, just the routine CRISPR / Cas12a method used to to detect P. cambivora.
(2)The resolution of the Figures are particularly unclear
(3)Figure 1,Schematic diagram of the RPA-CRISPR / Cas12a assay, the Pattern Diagram of RPA-CRISPR / Cas12a assay can be seen elsewhere, and There is no need for an independent figure.
(3) Numerous formatting errors and scientific writing issues
Line 135: results were obtained using the CRISPR / Cas12a system within 30 min.., two ..
Line 151: 2 μL g DNA
Line 195: Nine mature mature
Line 230: The optimal cutting time of Cas12 a
Line 264: was 10 pg·μL-1 (Figure 5A, B).
Author Response
1. The major problem was the author conclude that a new detection method based on RPA- CRISPR / Cas12a was established to detect P. cambivora, actually, it was not a new method, just the routine CRISPR / Cas12a method used to to detect P. cambivora.
Response : We thank the reviewer for this comment. Our main innovation is to apply this detection method to the detection of P. cambivora, and to screen its new specific target for detection. And I will explain the text that the technology has been modified for the part of the new technology.
2. The resolution of the Figures are particularly unclear
Response : We thank the reviewer for this comment. I have changed the clarity of the images in the article according to the journal's requirements (minimum 1000 pixels width/height, or a resolution of 300 dpi or higher).
3. Figure 1,Schematic diagram of the RPA-CRISPR / Cas12a assay, the Pattern Diagram of RPA-CRISPR / Cas12a assay can be seen elsewhere, and There is no need for an independent figure.
Response : We thank the reviewer for this comment. Because I belong to the same research group as similar authors and use the same mapping tools, there are similarities. I have put the mapping page in the text, which is lacking in other articles. I thought that perhaps adding a schematic diagram of the experiment to the text would more visually present the entire experiment to the reader. So I adjusted Figure 1 and chose to retain.
4. Numerous formatting errors and scientific writing issues
Line 135: results were obtained using the CRISPR / Cas12a system within 30 min.., two ..
Line 151: 2 μL g DNA
Line 195: Nine mature mature
Line 230: The optimal cutting time of Cas12 a
Line 264: was 10 pg·μL-1 (Figure 5A, B).
Response : We thank the reviewer for this comment. We thank the reviewers for their comments on this. This part of the error has been changed according to your requirements, please see the text.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Dear Authors,
Please find below my comments and suggestions:
Line 113: Remove ‘Please check if it could be rewritten as’.
Line 117: Insert a period ‘.’ before ‘The selected’.
Line 132: Remove ‘S’.
Line 281: I suggest removing the word ‘China’ after ‘Taiwan’ to maintain a neutral and non-political stance, which is essential in scientific papers.
Section 3.1 (lines 200-203): More than 1000 genes were identified as putative species-specific genes. Could you please describe the functional annotation of these genes? Additionally, please provide a detailed description of the criteria and rationale used to select only 10 genes from this larger set for designing the RPA primers.
Lines 326-327: Please correct this sentence: 'A new target gene of Phytophthora cambivora, g2339, was identified from the genome sequences of Phytophthora, Phytopythium, and Pythium…'. The gene g2339 is unique to Phytophthora cambivora according to your described procedure.
Author Response
Response to Reviewer 1 Comments
Dear reviewer:
We thank you for the additional comments. We revised the manuscript accordingly and the comment is attached below. We appreciate your work and look forward to hearing from you.
Sincerely,
Dr. Tingting Dai
Point：
- Line 113: Remove ‘Please check if it could be rewritten as’.
Response 1: Thanks for your positive comments and constructive suggestions. This sentence has been deleted.
- Line 117: Insert a period ‘.’ before ‘The selected’.
Response 2: Thanks for your positive comments and constructive suggestions. The error has been deleted, and see the latest version of the upload.
- Line 132: Remove ‘S’.
Response 3: Thanks for your positive comments and constructive suggestions. The error has been deleted.
- Line 281: I suggest removing the word ‘China’ after ‘Taiwan’ to maintain a neutral and non-political stance, which is essential in scientific papers.
Response 4: Thanks for your positive comments and constructive suggestions. I have modified Taiwan, China to Taiwan.
- Section 3.1 (lines 200-203): More than 1000 genes were identified as putative species-specific genes. Could you please describe the functional annotation of these genes? Additionally, please provide a detailed description of the criteria and rationale used to select only 10 genes from this larger set for designing the RPA primers.
Response5: Thanks for your positive comments and constructive suggestions.
The genome sequence of Phytophthora cambivora and (https://www.ncbi.nlm.nih.gov/genome/?term=Phytophthora, https://www.ncbi.nlm.nih.gov/genome/?term=Pythium, https://www.ncbi.nlm.nih.gov/genome/?term=Phytopythium) were compared and analyzed (e-value cutoff : 1e-5) to find the specific region of P. cambivora. According to the specific sequence of P. cambivora, more than 1000 genes (specific region) of P. cambivora with extremely low similarity to the gene sequences of other oomycetes were obtained, and then the most conserved genes in the top ten were selected from more than 1000 genes. Primer Premier 6.0 was used to design primers (primer length: 18-22 bp; the primer sequence should avoid three repeated bases connected; G + C content of 40-60 % is appropriate; the length of the amplified sequence is 200-300 bp).
- Lines 326-327: Please correct this sentence: 'A new target gene of Phytophthora cambivora, g2339, was identified from the genome sequences of Phytophthora, Phytopythium, and Pythium…'. The gene g2339 is unique to Phytophthora cambivora according to your described procedure.
Response 6: Thanks for your positive comments and constructive suggestions. This sentence has been modified, and see the latest version of the upload.
Author Response File: Author Response.docx
Reviewer 2 Report
The manuscript has been carefully revised an can be accepted in current form.
Author Response
Response to Reviewer 2 Comments
Dear reviewer:
Thank you for your response. We have made minor revisions to the manuscript based on the comments of another reviewer, and thank you again for your valuable review of this article.
Sincerely,
Dr. Tingting Dai
Author Response File: Author Response.docx
