-->PONE-D-25-60550-->-->Thermal optimized PCR coupled to CRISPR-Cas12a for rapid detection of blaOXA-1 resistance gene-->-->PLOS One

Dear Dr. Alcántara,

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.

============================== -->-->The reviewer has pointed out a number of areas in which revision is needed. Please addess all reviewer comments point by point.-->-->==============================

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Iddya Karunasagar

Academic Editor

PLOS One

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Reviewer's Responses to Questions

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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-->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: Thermal optimized PCR coupled to CRISPR-1 Cas12a for rapid detection of blaOXA-1 resistance gene

This study developed a CRISPR-Cas12a-based fluorescence method for the detection of blaOXA-1 gene, with a sensitivity of 8 CFU. Primers designed from consensus sequences of blaOXA-1 were employed in conventional PCR for initial optimization of conditions, followed by CRISPR-Cas detection.

Major comments

The study does not convincingly prove the specificity of the assay. The authors should have tested their primers with diverse blaOXA-1-harbouring isolates, both E. coli and other Enterobacterales, and other ESBL- and MBL-encoding genes to prove the specificity of the method developed.

What is the specific advantage of this method, when PCR methods for blaOXA variants are already available?

Further, the authors need to clearly define the goal (s) and the application of their work in the abstract and in the introduction section. It is only evident after section 2.4.3. (Spike-in pilot test) that the method was indeed designed to detect the target organisms (E. coli carrying blaOXA-1 gene) in chicken feces.

In L217-223: It is stated here that the mismatches in primer binding sites might potentially affect detection of the variants of blaOXA gene. This being the case, is this method specific to only blaOXA-1 gene? Does this not limit the application of this method, since blaOXA genes are very diverse?

L165: Here, amplification product of 564 bp has been mentioned. What does this product refer to?

5 x 10-3 attomoles of pure DNA: Please compare and discuss the sensitivity with pure DNA and the spiked bacterial cells (8 CFU/reaction). Alternatively, this may be represented as gene copies.

Minor comments

Please label A & B in Figure 2

Figure 3. Please move (A) to the next line

Reviewer #2: The study is simple yet performed with relevant outcomes. However, authors can address the following comments

1. What is the inclusion/exclusion criteria used while selecting 46 blaOXA-1 gene sequences?

2. Authors need to specify the version of tools used and parameter settings wherever applicable throughout the methodology

3. Times reported for different TRR with lab-made Taq (e.g., 0.6 °C/s = 83 minutes) differ from those with DreamTaq. Can authors clarify why total run times differ, given similar cycle numbers and temperatures?

4. Can authors provide alternative gel image for Figure 2A since the bands appear at different size range (could be due to imaging error)

5. Two different plate readers were used. Were the instruments cross-calibrated to ensure comparable fluorescence readings?

6. How was 10 blaOXA-1 negative E. coli isolates confirmed? Whether by PCR or WGS?

7. Authors used a 564 bp purified amplification product as target for Target concentration curve. Why is this length different from the 282 bp region described earlier?

8. For amplification with lab-made enzyme cycle time was 10 s per step, while 1 s protocol was followed for DreamTaq. Can authors justify the difference?

9. During spike test, OD600 was adjusted to 0.05 before serial dilutions. Was CFU conversion calibrated for this isolate or assumed from literature?

10. “From each selected dilution, 100 μL were transferred to 900 μL of the fecal suspension, then, 250 μL of each suspension was used for total DNA extraction, corresponding to an estimated 8 and 60 total CFUs, respectively.” Was extraction efficiency (recovery of DNA per CFU) evaluated or assumed?

11. “Extraction was performed using the ZymoBIOMICS™ DNA Miniprep Kit (Cat. N° D4300, Zymo Research), following the manufacturer's protocol.” Could authors compare kit-based extraction to simpler or cheaper methods that may be relevant for field deployment?

12. For spiked samples, authors used the modified PCR with DreamTaq and subsequent CRISPR detection. Did authors also test lab-produced Taq in the fecal matrix, and if not, why?

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Reviewer #1: No

Reviewer #2: No

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-->PONE-D-25-60550R1-->-->Thermal optimized PCR coupled to CRISPR-Cas12a for rapid detection of blaOXA-1 resistance gene-->-->PLOS One

Dear Dr. Alcántara,

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.

==============================

Some minor changes in language improvement, formattig needed

==============================

Please submit your revised manuscript by May 23 2026 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 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Iddya Karunasagar

Academic Editor

PLOS One

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Additional Editor Comments :

Please see some comments from reviewer regarding improvements needed.

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-->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: Yes

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

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-->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: The authors have responded to the queries raised in the previous review. Yet, the application of blaOXA-1 group -specific detection method has limited applicability in view of the fact that this does not cover the most common blaOXA gene(s) either in E. coli or other Gram-negative pathogens such as Acinetobacter baumannii.

The application of an in-house polymerase is something unique in this study, although it is not clear how the results obtained using an in-house polymerase can be reproduced in other laboratories beyond the geographic region where this study was performed. Further, the authors have stated in the manuscript that this polymerase had a lower efficiency (processivity) compared to commercial polymerase (s). In my opinion, the section describing in-house polymerase may be removed, as the data/results are relevant only to the laboratory employing it.

A few suggestions are below for authors’ consideration

75: Please correct as ..”needing long amplification times.

L96-97: Please rephrase this sentence.

Please write blaOXA correctly throughout the manuscript. OXA should be subscript of bla and in non-italics.

L133: The dNTP concentration of 0.5 nM seems to be too low. Please check. Similarly, the Taq concentration is generally stated in units, but you have indicated in ng/µL.

L163-164: Please write bla genes in correct format.

L208: Please provide the strain number/designation of blaOXA-1 isolate you used in this study

L208: Replace “cultivated” with “grown”

L211: Seeded? It appears that you performed a spread plate. However, spread plate method employs 100 microliters, not 50.

L214-216; Please rephrase the sentence.

L446-451: Agreed that qPCR and NGS are expensive. In what way your method is less expensive or technically less demanding than conventional PCR?. This method also requires PCR amplification, followed by fluorescence detection.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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-->

Thermal optimized PCR coupled to CRISPR-Cas12a for rapid detection of blaOXA-1 resistance gene

PONE-D-25-60550R2

Dear Dr. Alcántara,

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.

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Kind regards,

Iddya Karunasagar

Academic Editor

PLOS One

Additional Editor Comments (optional):

All reviewer comments have been addressed.

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

**********

-->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

**********

-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: 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

**********

-->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

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-->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: The authors have satisfactorily addressed all the queries raised in the previous review.

L263-265 & ub Figure legends: Italicize "bla".

Fig 2B legend: "Same as A, using...

Fig. 3B: the x axis lebel say "target log1o attomoles, but the numbers are represented to the power of 10. Please check if this is correct.

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Reviewer #1: No

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