Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of Shigella spp. and enteroinvasive Escherichia coli

Zheng Bian; Wenbo Liu; Junhua Jin; Yanling Hao; Linshu Jiang; Yuanhong Xie; Hongxing Zhang

doi:10.1371/journal.pone.0278869

Peer Review History

Original SubmissionAugust 16, 2022
30 Sep 2022 Decision Letter - Iddya Karunasagar, Editor PONE-D-22-22928Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of the bacterium Shigellaspp. and enteroinvasive Escherichia coliPLOS ONE Dear Dr. Zhang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please provide clarifications and revise addressing reviwer comments. Please submit your revised manuscript by Nov 14 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Additional Editor Comments: Please see the reviewer comments and provide clarifications and revision addressing all comments point by point. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have developed an RPA-LFD for rapid detection of Shigella spp. and enteroinvasive Escherichia coli using bacterial pure culture. The optimized assay cannot differentiate Shigella spp. and enteroinvasive Escherichia coli, and they cross-reacted with each other; Hence the title should be revised accordingly. Also, this can be discussed in the discussion section. What was the rationale for using the ipaH gene? Line: 81-82: mention the details of the laboratory from where bacterial strains were procured Table 1: typhimurium can be written as “Typhimurium” and non-italicize Line 101: mention a reference for this “phenol-chloroform method” Reviewer #2: PONE-D-22-22928 Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of the bacterium Shigella spp. and enteroinvasive Escherichia coli This study developed a recombinase polymerase-based assay followed by lateral flow dipstick (RPA-LFD) detection of Shigella spp. and the enteroinvasive E. coli. The ipaH gene common to both the organisms was used as the target for RPA. This naked eye detection method is rapid and specific to the target organisms. Major comments: Although the study successfully developed RPA-LFD method, its utility has not been demonstrated beyond identifying pure cultures of bacteria. Precisely, where this assay can be employed? For clinical specimens, foods or water?. The performance of the assay can vary in different matrices. In this context, although the study has accomplished developing a RPA-LFD method for Shigella and EIEC, it has not demonstrated it applicability to any sample type. Apart from the sample matrix, the DNA extraction method also has bearing on the sensitivity of the assay. The specificity of the assay can also vary depending on the background microbiota, which again can vary across the the sample types. It is necessary that the specificity of the assay is established using the natural samples. The sensitivity of RPA-LFT was determined using the plasmid containing cloned fragment of ipaH gene. Ideally, this should have been done using 10-fold serially diluted bacterial cultures (Shigella and EIEC), followed by DNA extraction. Minor comments Title: Consider removing “the bacterium” from the title L40: “pathogens”. L45-46: Please provide references to this statement. L50: Please replace “abundant” with “present” Table 1: Please expand CICC in the foot note, and the source/origin L95: Please clarify what did you mean by “NCBI was used to design primers” L99-100: “After mixing, 2.5 µL of Mg(Ac)2 (280mM) was added to start the reaction”. L109: Please delete “Most of..” L178-180: Please rephrase this sentence to convey the correct interpretation ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: PONE-D-22-22928_review.docx https://doi.org/10.1371/journal.pone.0278869.r001
Revision 1
18 Oct 2022 Author Response Reviewer: 1 Q1: The optimized assay cannot differentiate Shigella spp. and enteroinvasive Escherichia coli, and they cross-reacted with each other; Hence the title should be revised accordingly. Also, this can be discused in the discussion section. A: The title has been modified according to your suggestions and discussed in the discussion. Q2: What was the rationale for using the ipaH gene? A: We focused on foodborne pathogens that can produce toxins in food, and the ipaH gene is one of the major genes encoding virulence factors in Shigella and enteroinvasive Escherichia coli. In addition, Hartman et al. [1] found that ipaH gene has multi-copy nature and exists on both chromosome and invasion plasmid, which makes it superior to other single-copy virulence genes. At the same time, Lampel and Orlandi [2] reported that storage before testing would lead to the loss of invasive plasmids. Molecular assays designed to detect single-copy genes on invasive plasmids may fail, so molecular assays targeting ipaH genes have a greater chance to detect invasive plasmids or chromosomes. Q3: Line: 81-82: mention the details of the laboratory from where bacterial strains were procured A: The manuscript has been revised according to your suggestion. Q4: Table 1: typhimurium can be written as “Typhimurium” and non-italicize A: The manuscript has been revised according to your suggestion. Q5: Line 101: mention a reference for this “phenol-chloroform method” A: The manuscript has been revised according to your suggestion. Reviewer: 2 Q1: The specificity of the assay can also vary depending on the background microbiota, which again can vary across the sample types. It is necessary that the specificity of the assay is established using the natural samples. A: The actual sample testing experiment has been supplemented according to your suggestion. Q2: The sensitivity of RPA-LFD was determined using the plasmid containing cloned fragment of ipaH gene. Ideally, this should have been done using 10-fold serially diluted bacterial cultures (Shigella and EIEC), followed by DNA extraction. A: Because the extraction method of DNA sample will also affect the sensitivity of detection, we use standard plasmid for 10-fold gradient dilution in order to pursue the accuracy of detection limit. In the actual sample inspection, in order to achieve the universality of detection, use 10-fold of continuously diluted bacterial cultures (Shigella and EIEC) according to your recommendations, and then extract DNA. Q3: Consider removing “the bacterium” from the title A: The title has been revised according to your suggestions Q4: L40: “pathogens”. A: The manuscript has been revised according to your suggestion. Q5: L45-46: Please provide references to this statement. A: The manuscript has been revised according to your suggestion. Q6: L50: Please replace “abundant” with “present” A: The manuscript has been revised according to your suggestion. Q7: Table 1: Please expand CICC in the foot note, and the source/origin A: The manuscript has been revised according to your suggestion. Q8: L95: Please clarify what did you mean by “NCBI was used to design primers” A: Primers were designed and screened using the primer-BLAST function of the National Center for Biotechnology Information (NCBI).And the manuscript has been revised. Q9: L99-100: “After mixing, 2.5 µL of Mg(Ac)2 (280mM) was added to start the reaction”. A: The manuscript has been revised according to your suggestion. Q10: L109: Please delete “Most of..” A: The manuscript has been revised according to your suggestion. Q11: L178-180: Please rephrase this sentence to convey the correct interpretation A: The manuscript has been revised according to your suggestion. Reference 1. Hartman, A. B., M. Venkatesan, E. V. Oaks, and J. M. Buysee. Sequence and molecular characterization of multicopy invasion plasmid antigen, ipaH of Shigella flexneri. J. Bacteriol. 1990; 172: 1905–1915. 2. Lampel, K. A., and P. A. Orlandi. Polymerase chain reaction detection of invasive Shigella and Salmonella enterica in food. Methods Mol. Biol. 2002; 179: 235–244. Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0278869.r002
18 Nov 2022 Decision Letter - Iddya Karunasagar, Editor PONE-D-22-22928R1Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of the ipaH gene from Shigellaspp. and enteroinvasive Escherichia coliPLOS ONE Dear Dr. Zhang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Please see the comment on title modification. ============================== Please submit your revised manuscript by Jan 02 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Iddya Karunasagar Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Please see the reviewers comment regarding modification of title to better reflect the contents of the paper. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: The authors have modified the manuscript sufficiently as suggested in my first review. However, the title might require modification. It says "....... detection of the ipaH gene from Shigella spp. and enteroinvasive Escherichia coli". Here you goal is not just to detect ipaH gene. Instead, you are using this gene as a marker to detect two species of bacteria. Therefore, the title can be "Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of Shigella spp. and 4 enteroinvasive Escherichia coli ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0278869.r003
Revision 2
19 Nov 2022 Author Response Dear reviewer: Thank you very much for your comments. We have revised our manuscript according to your suggestions. And the detail information is following. Sincerely, Yuanhong Xie Reviewer: 2 Q1: However, the title might require modification. It says "....... detection of the ipaH gene from Shigella spp. and enteroinvasive Escherichia coli". Here you goal is not just to detect ipaH gene. Instead, you are using this gene as a marker to detect two species of bacteria. Therefore, the title can be "Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of Shigella spp. and enteroinvasive Escherichia coli A: The title has been modified according to your suggestions. Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0278869.r004
28 Nov 2022 Decision Letter - Iddya Karunasagar, Editor Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of Shigellaspp. and enteroinvasive Escherichia coli PONE-D-22-22928R2 Dear Dr. Zhang, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Iddya Karunasagar Academic Editor PLOS ONE Additional Editor Comments (optional): All reviewer comments have been addressed. Reviewers' comments: https://doi.org/10.1371/journal.pone.0278869.r005
Formally Accepted
1 Dec 2022 Acceptance Letter - Iddya Karunasagar, Editor PONE-D-22-22928R2 Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of Shigella spp. and enteroinvasive Escherichia coli Dear Dr. Zhang: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Iddya Karunasagar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0278869.r006

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