Direct PCR for Rapid and Safe Pathogen Detection: Laboratory Evaluation Supporting Field Use in Infectious Disease Outbreak

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper “Direct PCR for Rapid and Safe Pathogen Detection: Field Testing in Emerging Infectious Disease Outbreaks?” evaluate evaluated DNA/RNA Defend Pro (DRDP) buffer, a novel viral-inactivating transport medium designed to stabilize nucleic acids and allow direct PCR without nucleic acid extraction. Some comment are stated below:

1. The process of primers design, and how did they were evaluated should be stated in
2. Specificity and repeatability should also be evaluated.
3. Key figures should be applied.
4. Some clinical samples should be supplied to evaluate the application of this method on clinical of environmental samples.

Author Response

Reviewer 1

Comment: “The process of primers design and how they were evaluated should be stated.”

Response: We agree and thank the reviewer for highlighting the need to clarify our primer design process. Our qPCR primer/probe sets were not designed de novo for this study; rather, we adopted previously published, validated primer/probe sequences for HSV-1, HSV-2, and VZV. Specifically, we have now stated in the Materials and Methods (PCR Assay and Conditions subsection) that the HSV-1 and HSV-2 assays use primers and probes from Watzinger et al. (2004) and Weidmann et al. (2003), and the VZV assay uses the primers/probe from Depledge et al. (2011).

Comment: “specificity and repeatability”: These primer–probe sets have been extensively validated in the literature and by us and have been part of our routine clinical diagnostic workflow (as validated Laboratory Developed Tests) for the past two decades. Their performance and quality have been continuously monitored under standards set by the College of American Pathologists (CAP) and the Bureau de Normalization du Québec (BNQ). To make this completely clear, we have added the following statement to the Methods section (new text in italic in the manuscript):

“The primers and probes used in this study were adapted from previously validated protocols: HSV-1 and HSV-2 assays from Watzinger et al. (2004) and Weidmann et al. (2003), and the VZV assay from Depledge et al. (2011) [14,15,16]. These primer/probe sets have been integrated into our routine clinical diagnostic workflow as validated Laboratory Developed Tests (LDTs) and have been consistently utilized in our laboratory for the past two decades. Their clinical performance and quality assurance have been continuously monitored and maintained in compliance with standards set by the College of American Pathologists (CAP) and the Bureau de Normalisation du Québec (BNQ).”

Comment 2 and 3: “Key figures (2) and clinical sample (3)”

Key figures are added (2 additional Tables (Table 3A and Table 4A in appendix) , making total of 4 tables). Graphical abstract was also added to reflect the advantages of direct PCR used with virus inactivation buffer, thus simplifying protocol and time of sample processing comparing to Universal Transportation Media.

This work presents laboratory evaluation of new DRDP medium and it is not a clinical trial. This evaluation uses a few anonymized positive sample/s as laboratory quality controls. The evaluation indicates that described protocol might be used in the case of emerging infection outbreak/s (DNA enveloped viruses and previously published RNA viruses) without compromising expected assay performance (specificity, sensitivity). Increase in biosafety, decrease in sample processing complexity and turnaround time was mentioned as applicable solution in a future clinical trial.

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors evaluated the performance of DNA/RNA Defend Pro (DRDP) buffer for direct PCR diagnostics in pathogen detection, using HSV-1, HSV-2, and VZV as model DNA viruses. DRDP demonstrated advantages over traditional universal transport medium (UTM), including enhanced biosafety (immediate pathogen inactivation), equivalent or improved sensitivity, and simplified workflows (no nucleic acid extraction or thermal lysis). While the data are robust and support the utility of DRDP in field-deployable diagnostics, critical issues require clarification. My recommendation is major revision, with detailed comments below:

1.The EDTA-induced inhibition was attributed to magnesium ion chelation, but no direct evidence (e.g., Mg²⁺ titration curves) was provided. Quantitative Mg²⁺ restoration experiments are required to validate this mechanism.

2.Although the DRDP technology claims to preserve samples at room temperature with stability, it lacks crucial long-term data support: specific storage duration, nucleic acid degradation rate, and changes in detection accuracy and other quantitative indicators are not provided, making it impossible to evaluate its ability to ensure long-term effectiveness in practical applications.

3.Only three DNA viruses, HSV-1, HSV-2, and VZV, were used as models, without involving other common pathogens such as RNA viruses. This limits the comprehensive evaluation of the application scope of DRDP, and it is difficult to determine its universality for the detection of different types of pathogens.

4.The formats of references are not uniform. Some references lack DOI or volume and issue numbers, and the positions of publication years are inconsistent. Please check carefully and standardize the reference format.

5.For the commercial PCR platform test (DiaSorin Simplexa), only the statement that "the Cq value is slightly lower" was mentioned, but no specific data or results of repeated experiments were shown, making the demonstration somewhat weak.

6.The expression "exempted by the ethics review committee" is ambiguous. To comply with the journal's policy, please clarify the name of the ethics committee, approval number, or exemption criteria.

7.The description of "anonymous remaining clinical samples" lacks detailed information (such as collection protocol, storage conditions). Please describe the sample processing procedure to ensure the transparency of the methodology.

Author Response

 

 

Reviewer 2

Comment 1: “EDTA-induced inhibition was attributed to magnesium ion chelation, but no direct evidence (e.g., Mg²⁺ titration curves) was provided. Quantitative Mg²⁺ restoration experiments are required.”

Response: Thank you for this important observation. We have performed experiments to address this point and repeated them. Specifically, we titrated magnesium supplementation to quantitatively evaluate its ability to reverse EDTA-induced PCR inhibition for both LDT and Commercial IVD Tests. The results are now included in the Results (Section 3.2) and summarized in a new Appendix Table A3 (for LDT) and A4 (for IVD test). In brief, adding 10 mM Mg²⁺ to reactions containing high fractions of DRDP buffer completely restored amplification (Cq values returned to baseline), whereas lower Mg²⁺ concentrations (e.g., 2.5 mM or 5 mM) resulted in only partial or no recovery of amplification. These findings confirm that the observed inhibition was indeed caused by EDTA chelation at high DRDP concentrations. We have added a description of these results to the Results section, and the supplementary Appendix now contains Table A3 and A4 detailing the Mg²⁺ titration outcomes.

To demonstrate that this solution (Mg supplementation) works in a commercial assay context, we evaluated DRDP on the Diasorin Simplexa HSV-1/2 & VZV Direct PCR platform. We found that PCR inhibition on the commercial platform could also be resolved by magnesium supplementation. This new data is presented in Appendix Table A4. The following new text was added to Section 3.4 of the Results:

“Detailed Mg²⁺ titration experiments assessing DRDP buffer with Mg²⁺ concentrations of 0 mM, 5 mM, 10 mM, 15 mM, and 20 mM are presented in Appendix A, Table A4. These experiments clearly demonstrate that equivalent Ct values (within one Ct unit of the UTM control Ct value) were restored beginning at approximately 10 mM Mg²⁺, with optimal, lowest Ct values consistently achieved at 15 mM and 20 mM Mg²⁺ concentrations.

These results confirm the compatibility of DRDP sample collection medium, achieving equivalent performance while enhancing biosafety, reducing turn-around-time and reducing operational complexity and corresponding cost (Figure 1).”

Comment 2: “Long-term stability data for DRDP technology at room temperature is insufficient.”

Response: We have added clarification and supporting references to address the sample stability concerns. In our study, we did not observe any shift in Cq values for samples stored in DRDP, even after multiple transfers between 4 °C and room temperature over a two-week period (to simulate routine laboratory handling). To strengthen this point, we have cited independent studies (including manufacturer data) demonstrating nucleic acid stability in DRDP and similar inactivation media at ambient temperatures. In the Discussion, we reference Claeys et al. (2024) and the InActiv Blue IFU (2024), which report enhanced stability of viral RNA/DNA in the DRDP buffer at room temperature. These additions to the manuscript (with new references [8,9] added) address the reviewer’s concern by providing evidence that DRDP can maintain nucleic acid integrity for extended periods without cold storage. The relevant clarifying text is underlined in the revised Discussion section.

Comment 3: “Only DNA viruses were evaluated; RNA viruses and other common pathogens were not included.”

Response: The focus of our work was on enveloped DNA viruses (HSV-1, HSV-2, VZV) in the context of biosafety and a specific diagnostic challenge (distinguishing herpesviruses from mpox lesions). We appreciate the importance of demonstrating DRDP’s applicability to RNA viruses and other pathogens, and we agree that our findings should be understood in a broader context. In fact, other groups have already tested DRDP or similar inactivation transport buffers with RNA viruses such as influenza, SARS-CoV-2, and RSV. Considering the reviewer’s comment, we have added a statement in the Discussion noting that our findings are consistent with those prior evaluations on RNA viruses. We cite recent studies showing that an inactivating transport medium can enable direct PCR for these RNA viruses without sensitivity loss. Our added text emphasizes that our results extend this direct-PCR approach to DNA viruses and that the DRDP method is broadly applicable. This new sentence (with relevant references) is included in the Discussion (underlined in the revised manuscript).

Additionally, in the Conclusions we now explicitly mention that ongoing and future studies will explore using DRDP for other pathogens and with field-deployable PCR platforms. For example, we state that:

“Ongoing and future studies extending this approach to other pathogens and integrating it with field-deployable PCR platforms will further validate DRDP’s role in advancing molecular diagnostics, while also protecting operators from exposure to potential pathogens.”

Comment 4: “Reference formats are inconsistent.”

Response: We apologize for the formatting issues. We have now reformatted all references in a consistent Vancouver style (numerical citations in order, with complete bibliographic details). The reference list in the revised manuscript has been thoroughly checked for consistency in punctuation, italics, journal abbreviations, et al. usage, etc. All in-text citation numbers have been updated to reflect the addition of new references and to maintain the correct order. For example, we added new references to support the points on sample stability and on prior evaluations with RNA viruses, and the numbering sequence was adjusted accordingly. The final manuscript’s References section is now uniform in style and numbering. We appreciate the reviewer noting this, and have ensured the issue is fully corrected.

Comment 5: “Specific data comparing commercial PCR platform (DiaSorin Simplexa) results were insufficient.”

Response: We acknowledge that our evaluation of the DiaSorin Simplexa HSV-1/2 & VZV Direct PCR platform was limited in scope (a qualitative analytical comparison on a small number of control samples). A comprehensive, prospective comparison with a commercial assay is beyond the scope of this proof-of-concept study. Nevertheless, we included a brief experiment to ensure that DRDP does not adversely affect the performance of a well-established commercial test. In the revised manuscript, we have removed the statement that previously suggested a “~1 Cq unit improvement” with DRDP, in order to avoid any over-interpretation of our preliminary data. Instead, we now simply conclude that DRDP performs equivalently to UTM on the commercial assay (i.e. no loss of sensitivity when using DRDP). The focus in the text is now on demonstrating compatibility with the commercial platform rather than implying any superiority. This change is aimed at preventing confusion, as the reviewer rightly requested. (These edits are reflected in Section 3.4 of the Results. The phrasing about an improvement has been deleted, and we have added the new data as Appendix Table A3 and A4 to document the comparison.)

 

 

Comment 6: (“Exempted by the ethics review committee” wording): “The expression 'exempted by the ethics review committee' is ambiguous.”

Response: We have rewritten the statement about ethics approval to clearly explain why formal ethics review was not required for this study. In the Institutional Review Board Statement of the revised manuscript, we now specify that our project was a laboratory quality improvement study using only anonymized leftover clinical samples, and therefore it did not involve human subjects research that would trigger an IRB review. We explicitly reference the applicable guideline (TCPS2 Article 2.5 in Canada) which permits such studies without formal REB approval, and we note that this was confirmed in the past by our hospital’s IRB policies. The revised text (see the end of the Conclusions/IRB Statement section) now reads:

“According to TCPS2 Article 2.5, such quality improvement initiatives are exempt from formal Research Ethics Board (REB) review.” This clarification should remove any ambiguity about the ethical considerations and make it clear that no human subjects’ approval was needed for this work.”

Comment 7. The description of "anonymous remaining clinical samples" lacks detailed information (such as collection protocol, storage conditions). Please describe the sample processing procedure to ensure the transparency of the methodology.

Response: As per our standard operating procedure (SOP), samples were collected by healthcare providers in clinical offices and then transferred directly to the Microbiology Laboratory at the Jewish General Hospital (JGH).

Sample Types and Collection Protocol: Lesions or vesicles were sampled using flocked swabs placed into Universal Transport Medium (UTM), typically 1.5 or 3 mL (e.g., Copan UTM-RT red-topped tubes containing 1.5 mL of UTM), or using standard swab transport tubes (e.g., BBL CultureSwab Collection and Transport System). Only swabs obtained from skin, mucocutaneous, or genital lesions/vesicles were accepted.

Transport and Storage Conditions: Samples were transported to the laboratory at room temperature, typically arriving within 24 hours of collection. Upon arrival at the laboratory, specimens were refrigerated at 4°C until processing, and processed within a maximum of 72 hours. Any deviations or necessary modifications to the described protocol required prior consultation and explicit approval from the microbiologist on call, who determined acceptance or rejection of the sample for testing.

Please note that purpose of this report is not clinical study of assay performance, but parallel analytical performance among samples which are collected, transported and stored under identical conditions but in different media/buffers (UTM vs DRDP). Details related to general sample collection/transport/storage were not reported since they do not have impact on final results.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors, 

The presented MS, 'Direct PCR for Rapid and Safe Pathogen Detection: Field Test- 2
ing in Emerging Infectious Disease Outbreaks?' is very informative and presents an innovative approach for application of PCR based solution in PoC setting. 

The MS is also presenting the working and application side of the DRDP buffer technique, which is a patented technology by the company. 

However, the MS is lacking in the data comparison and allowing the readers to find more Insite with this approach. Author is suggested to add a table of comparison where they can clearly mention the suitability and advantages of the presented technique over the other research. 

A graphical abstract of introductory diagram is also good to have better understanding for the young researchers. 

Author suggested that it can be applied to emergency and pathogenic outbreak. did they make any rapid testing and validation of the approach?

 

Author Response

 

Reviewer 3

Comment 1: “The manuscript lacks data comparison tables and graphical abstracts.”

Response: We appreciate this valuable suggestion. In the revised manuscript, we have included additional data tables to present the PCR result comparisons more clearly. In total, there are now six tables (Tables 1 and 2 in the main text, and four more tables labeled A1–A4 in Appendix A) that comprehensively summarize the findings and comparisons discussed in the manuscript.

Moreover, as recommended, we created a Graphical Abstract to illustrate the workflow differences and benefits of using DRDP versus a traditional transport medium. We have added a caption for this Graphical Abstract after the main Abstract in the manuscript (the graphical figure itself will be provided separately as per journal guidelines, with the caption included in the text). The graphical abstract visually highlights DRDP’s advantages, such as immediate pathogen inactivation, the elimination of an extraction step, and streamlined processing, in contrast to a conventional medium. Both the addition of the new tables and the inclusion of the graphical abstract are clearly indicated in the revised manuscript. We believe these additions substantially improve the clarity and accessibility of our results.

Comment 2: “Were rapid testing and validation performed for emergency or pathogenic outbreaks?”

Response: Thank you for your valuable and critical question. Our study focused on laboratory work to demonstrate the utility of DRDP for routine diagnostics and foreseeable outbreak scenarios (such as a new variant of an enveloped virus, e.g. influenza or coronavirus, or the mpox situation described in the manuscript). Following this comment, we changed the title of manuscript: “Direct PCR for Rapid and Safe Pathogen Detection: Laboratory Evaluation Supporting Field Use in Infectious Disease Outbreak” clearly stated that this work presents Laboratory Evaluation. We did not have the opportunity to implement or validate DRDP during an active emergent outbreak or on a completely novel pathogen. We acknowledge that rapid validation of new methods during emergency outbreaks is extremely important; however, doing so for a completely unknown pathogen (of an unknown type) was beyond our current scope and resources. Such validation would require significant coordination with public health authorities and additional biosafety considerations.

That said, we envision that DRDP could be extremely useful in future outbreaks caused by new variants or subtypes of known enveloped viruses. For example, if a novel strain of a known virus (such as influenza, coronavirus, RSV, or a new herpesvirus strain) emerges, the fundamental DRDP method (immediate virus inactivation and direct PCR) would still apply and could greatly facilitate safe and rapid diagnostics. In the case of an entirely unknown pathogen (e.g., a completely new virus or other organism), additional assay development would of course be required before DRDP could be applied.

As suggested, we have made major changes to the manuscript title, indicting that we performed Laboratory evaluation supporting filed use of DRDP medium. Title covers potential outbreaks of new variants of known viral families (like influenza, coronavirus, RSV, herpesviruses, mpox). In the Conclusion of the revised manuscript, we now explicitly note that ongoing studies will extend DRDP to other pathogens, which aligns with the spirit of this comment. The current study establishes a proof-of-concept that will underpin such emergency validations when the opportunity arises. In summary, our DRDP strategy is already ready for clinical validation on the enveloped viruses tested by Manufacturer, and it can provide significant improvements in diagnostic speed, operational efficiency, and public health cost savings in outbreak settings.

 

 

Reviewer 4 Report

Comments and Suggestions for Authors

The article submitted by Brukner and Oughton, entitled "Direct PCR for Rapid and Safe Pathogen Detection: Field Testing in Emerging Infectious Disease Outbreaks?" addresses an important aspect of molecular diagnostic, which is DNA/RNA preparation from complex matrices. The authors compare the DNA/RNA Defend Pro buffer (DRDP) to the Universal Transport Medium (UTM) and demonstrate that their perfomance in direct quantitative PCR are similar, for double stranded DNA virus detection. Furthermore, they show that using DRDP allows skipping the denaturation step. The article is well written, the results are clear and convincing. It therefore deserves publication.

Major comment :

The authors use herpes virus HSV-1 and HSV2, and varicella zoster virus VZV. For herpes viruses, the viral load is not usually monitored during clinical management and for varicella zoster virus, the viral load is usually higher than 1000 copies/mL. However, for other viruses, such as HIV, viral loads are detected down to 50 copies/mL. Standard techniques that purify genetic materials (typically using silica beads and guanidium chloride) still have the advantage of concentrating the nucleic acid before its amplification. This is a limit of direct PCR. I suggest that the authors add a sentence explaining that direct PCR in DRDC has a sensitivity limit imposed by the volume used in the assay.

Minor comments :

• The question mark at the end of the title is not necessary
• P3, L107 : is magnesium added after the lysis or at the same time ?
• P4, Table 2 : the third column is "Cq shift"

Author Response

 

Reviewer 4

Comment 1: “Clarify that direct PCR in DRDP has a sensitivity limit imposed by assay volume.”

Response: We agree that this is an important caveat to mention. We have added a clarifying statement to the Discussion/Conclusion to explicitly note that direct PCR approaches (including ours) have an inherent sensitivity limitation due to the limited volume of inactivated sample that can be added to a PCR reaction. In other words, if a sample has an extremely low viral load, direct PCR (without an extraction/concentration step) might not detect it because one cannot simply add unlimited sample volume to the reaction. This new cautionary text is in italic in the revised Conclusion section. It emphasizes that while DRDP-enabled direct PCR is powerful for speed and safety, it may not be suitable for situations demanding the absolute lowest limit of detection. We believe this addition addresses the reviewer’s concern by tempering our conclusions and clearly informing readers of this practical effects. The exact wording added to the Conclusion is:

“It is important to note that direct PCR sensitivity has inherent limitations imposed by the sample reaction volume, potentially limiting detection in cases with an extremely low viral load. Consequently, when swab is collected in a large (1-3 mL) buffer volume the sensitivity might be reduced for some assays. On the contrary, although not tested here, a swab exposed to or impregnated with a small volume of buffer (50-100 µL) could potentially increase the analytical sensitivity dramatically in a direct PCR approach.”

Comment 2: “Minor comments on title punctuation, table labeling, and timing of magnesium supplementation.”

Response: We have made all the minor corrections as suggested by the reviewer. Specifically:

• Title punctuation: We removed the question mark from the title. The title now reads as a statement: “Direct PCR for Rapid and Safe Pathogen Detection: Field Testing in Emerging Infectious Disease Outbreaks” (without a question mark). We agree that the question mark was not necessary, and its removal makes the title more straightforward.
• Magnesium supplementation timing: We have clarified in the Methods (Section 2.2) when the magnesium was added during sample processing. The revised text now specifies that magnesium was supplemented immediately after sample lysis (within about 1–2 minutes of mixing the sample with the DRDP buffer). Introducing the extra Mg²⁺ almost immediately ensures that it chelates any EDTA before the PCR begins. (Revised text example: “Magnesium supplementation was performed in DRDP buffer post-lysis (within approximately 1–2 minutes of adding DRDP buffer to the sample) and around 5 minutes before adding it into PCR master mix).”
• Table labeling: We corrected the column label in Table 2 to “Cq shift” (replacing the previous incorrect label). Table 2 now clearly shows a column for the Cq shift (in cycles). We also double-checked all table titles, headings, and labels for accuracy and consistency throughout the manuscript.

These edits have all been implemented in the revised manuscript. (Because they were straightforward fixes, we did not specifically underline the removal of the question mark in the title or the single-word change in the table label.) We appreciate the reviewer’s careful attention to these details, which has helped us improve the manuscript’s clarity and accuracy.

 

 

Reviewer 5 Report

Comments and Suggestions for Authors

The study described in the manuscript “Direct PCR for Rapid and Safe Pathogen Detection: Field Testing in Emerging Infectious Disease Outbreaks?” is sound but does not consider the title regarding the application of tested buffer during emerging infectious disease outbreaks. On the onset of such outbreaks, the pathogen is often not known or no virus stock is available. For the application at that time, other sample material has to be tested additionally to the virus stocks already tested here, for example swabs, tissue samples, biofluids. This is the most important point of my concern with this manuscript. Such tests can be made up fast and the results can be easily included in the existing manuscript. I would also like to see the results for other viruses than DNA viruses, but this might be beyond the scope of this manuscript.
Regarding methods, did the authors skip a nucleic acid extraction for both buffer systems (DRDP and UTM) (see line 84f)? Would be interesting to see the results of other sample materials. Actually, a comparison should be made with extracted samples to know which virus amount could be lost when skipping the extraction step (although this procedure is recommended by the company). This is also an important point.
Regarding results, I would appreciate when the authors would show the results that are explained in sections 3.4 and 3.5 also in tables or graphs.
Discussion, line 229f; “ambient temperature storage” was not tested for longer duration.
Discussion, line 236; “reliability” was not tested in the study.

Author Response

Reviewer 5

Comment 1: “Title does not reflect the buffer application in outbreaks involving unknown pathogens.”

Response: We understand the concern raised about the title potentially implying a broader scope than the study covers. The title of our manuscript is changed, clearly stating our work as “laboratory evaluation”. New title is:

“Direct PCR for Rapid and Safe Pathogen Detection: Laboratory Evaluation Supporting Field Use in Infectious Disease Outbreak,”

It is intended to convey that the DRDP method can be applied in outbreak situations. We did not use the word “unknown” in the title or text; rather, our focus was on known classes of pathogens (specifically enveloped viruses) that could cause new outbreaks – for example, a new strain of a known virus. We acknowledge that validating DRDP on completely novel pathogens (of entirely unknown identity) is beyond what we have done in this study. However, we believe the current title is still appropriate because it emphasizes our laboratory evaluations in the context of outbreak readiness without explicitly claiming validation on unknown agents.

To address the reviewer’s point, changed the title (aside from the punctuation fix noted by Reviewer 4) because it highlights the main application (rapid, safe diagnostics in outbreak scenarios) and we clarify in the manuscript that our examples are within known virus families. In our Discussion and Conclusion, we also stress that future work could extend this approach to other pathogens, which implies how it might be used when new diseases emerge. In order to avoid making it title overly narrow (for example, listing specific viruses in the title would unintentionally exclude others), we use term “infectious disease outbreak”.

In summary, we appreciate the perspective that completely “unknown” pathogen outbreaks pose additional challenges. We note that any major outbreak likely to occur soon, as history suggests, will most probably be caused by variants of known organisms (e.g., a novel influenza or coronavirus), for which our method is directly relevant. 

Comment 2 Regarding methods, did the authors skip a nucleic acid extraction for both buffer systems (DRDP and UTM) (see line 84f)?

Response: Yes, that is clearly stated now in all sections of MS: “Materials and Methods: evaluating DRDP vs UTM for extraction-free PCR in a field context” and later in MM section “No nucleic acid extraction was performed on any samples.”

Comment 3 …virus amount could be lost when skipping the extraction step (although this procedure is recommended by the company) …

Response: The question of direct PCR versus Nucleic Acid Extraction is additionally clarified and cited. We also added the following text in Conclusion:

 “It is important to note that direct PCR sensitivity has inherent limitations imposed by the sample reaction volume, potentially limiting detection in cases with an extremely low viral load. Consequently, when swab is collected in a large (1-3 mL) buffer volume the sensitivity might be an issue, for some assays. On the contrary, although not tested here, a swab exposed to, or impregnated with a small volume of buffer (50-100 µL) could potentially increase the analytical sensitivity dramatically in a direct PCR approach.”

Comment 4 Regarding results, I would appreciate when the authors would show the results that are explained in sections 3.4 and 3.5 also in tables or graphs.

Discussion, line 229f; “ambient temperature storage” was not tested for longer duration.

Discussion, line 236; “reliability” was not tested in the study.

Response: Thank you for the suggestion. The results were added in the form of Tab 3A and Tab 4A, in the appendix of Revised manuscript.

The stability and reliability of DRDP was cited as previously peer review information and manufacturer claims.

 

Additional Note: All of the above changes have been incorporated into the revised manuscript. The manuscript has also been re-formatted to adhere to the journal’s guidelines. We sincerely thank the reviewers and the editors for their valuable comments, which have helped us improve the clarity, accuracy, and rigor of our manuscript. Please let us know if any further adjustments are required.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

NA

Author Response

25 June 2025
Montreal, QC, Canada

Dear Editor and Reviewers,

On behalf of all co-authors, I would like to extend our sincere thanks for your time, careful evaluation, and constructive comments throughout the peer review process of our manuscript, “Direct PCR for Rapid and Safe Pathogen Detection: Field Testing in Emerging Infectious Disease Outbreaks.”

We deeply appreciate the insightful feedback provided during the review, which has helped us improve the clarity, structure, and scientific rigor of the manuscript. The reviewers' observations and recommendations led us to refine our presentation, expand the discussion of key findings, and better articulate the implications of our work.

We are grateful for your support and are pleased that the manuscript has now been accepted for publication in LabMed. We hope it will contribute meaningfully to the ongoing dialogue around rapid, decentralized diagnostic strategies and serve as a useful resource for both research and clinical communities.

Thank you again for your continued guidance and for facilitating a very constructive and collegial review experience.

With best regards,
Ivan Brukner, PhD
OPTILAB – QC, Canada; Lady Davis Institute; McGill University
Editorial Board Member, LabMed MDPI
✉ ivan.brukner2@mcgill.ca

Matthew Oughton, MD
Jewish General Hospital, McGill University Health Centre (MUHC), Montreal, QC
✉ matthew.oughton@mcgill.ca

Reviewer 2 Report

Comments and Suggestions for Authors

All the comments were replied, this paper could be accepted.

Author Response

25 June 2025
Montreal, QC, Canada

Dear Editor and Reviewers,

On behalf of all co-authors, I would like to extend our sincere thanks for your time, careful evaluation, and constructive comments throughout the peer review process of our manuscript, “Direct PCR for Rapid and Safe Pathogen Detection: Field Testing in Emerging Infectious Disease Outbreaks.”

We deeply appreciate the insightful feedback provided during the review, which has helped us improve the clarity, structure, and scientific rigor of the manuscript. The reviewers' observations and recommendations led us to refine our presentation, expand the discussion of key findings, and better articulate the implications of our work.

We are grateful for your support and are pleased that the manuscript has now been accepted for publication in LabMed. We hope it will contribute meaningfully to the ongoing dialogue around rapid, decentralized diagnostic strategies and serve as a useful resource for both research and clinical communities.

Thank you again for your continued guidance and for facilitating a very constructive and collegial review experience.

With best regards,
Ivan Brukner, PhD
OPTILAB – QC, Canada; Lady Davis Institute; McGill University
Editorial Board Member, LabMed MDPI
✉ ivan.brukner2@mcgill.ca

Matthew Oughton, MD
Jewish General Hospital, McGill University Health Centre (MUHC), Montreal, QC
✉ matthew.oughton@mcgill.ca

Reviewer 5 Report

Comments and Suggestions for Authors

okay

Author Response

25 June 2025
Montreal, QC, Canada

Dear Editor and Reviewers,

On behalf of all co-authors, I would like to extend our sincere thanks for your time, careful evaluation, and constructive comments throughout the peer review process of our manuscript, “Direct PCR for Rapid and Safe Pathogen Detection: Field Testing in Emerging Infectious Disease Outbreaks.”

We deeply appreciate the insightful feedback provided during the review, which has helped us improve the clarity, structure, and scientific rigor of the manuscript. The reviewers' observations and recommendations led us to refine our presentation, expand the discussion of key findings, and better articulate the implications of our work.

We are grateful for your support and are pleased that the manuscript has now been accepted for publication in LabMed. We hope it will contribute meaningfully to the ongoing dialogue around rapid, decentralized diagnostic strategies and serve as a useful resource for both research and clinical communities.

Thank you again for your continued guidance and for facilitating a very constructive and collegial review experience.

With best regards,
Ivan Brukner, PhD
OPTILAB – QC, Canada; Lady Davis Institute; McGill University
Editorial Board Member, LabMed MDPI
✉ ivan.brukner2@mcgill.ca

Matthew Oughton, MD
Jewish General Hospital, McGill University Health Centre (MUHC), Montreal, QC
✉ matthew.oughton@mcgill.ca