Establishment and Application of a SYBR Green I qPCR Detection Method Based on the CP40 Gene of Corynebacterium pseudotuberculosis Biovar Ovi

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

            The presented manuscript is dedicated to design and validation of a novel PCR-based test for detection of Corynebacterium pseudotuberculosis biovar ovi, a pathogen of small ruminants that significantly decreases economic yield of sheep and goat farming. The suggested method utilizes qPCR with an intercalating dye for discrimination of the targeted biovar among other bacteria infecting ruminants. While the study’s concept itself is valid and timely, a number of questions needs to be cleared before possible publication. Please find them below.

1. Not all scientific names are italicized.
2. Authors are requested to provide more information about primer design, specifically, alignments demonstrating absence of mismatches under primers and the number of aligned genomes. The same can be said about alignment with of different biovars, because this is crucial for specificity of NAATs.
3. Rpm is used instead of rcf.
4. 3.2. Traditional PCR amplification and 2.4.2. Optimization of reaction conditions — important details are missing including reaction volume and reagents concentrations.
5. Page 4, line 157: “in section 1.3.3.” — it seems that the actual number is different from the given one.
6. 4.4. Specific test — what were DNA concentrations?
7. 4.5. Sensitivity test — how many technical repeats were analyzed and how was the LoD calculated?
8. 4.7. Clinical sample testing — a brief description of the purification protocol is necessary. “After centrifugation at 4000 rpm for 20 minutes, viral genomic RNA was extracted from the supernatant using a viral DNA/RNA extraction kit. Genomic nucleic acid was removed prior to reverse transcription into cDNA for subsequent analysis.” — why were gDNA discarded if it was actually targeted by the designed assay?
9. 1. Amplification results of target genes — what sample was used for the amplification? Also, Figure 1 does not represent the PCR results as it mentioned in the text.
10. 4. Optimization of SYBR Green I qPCR reaction system — the results of optimization experiments would also be interesting, especially, a gradient of annealing temperature.
11. 9. Clinical specimen validation — what were Cq values of discordant samples? Also, the analysis would be more robust, if the samples were tested by an independent previously reported and commonly using method.
12. Authors are requested to compare characteristics of their test with previously published and discuss possible limitations of this approach. Also, it is not directly stated why an intercalating dye was used instead of TaqMan or other similar probes. The latter approach is more specific and less prone to false-positive results due to non-specific amplification of primer-dimes and other non-targeted DNA sequences.

Author Response

1. Not all scientific names are italicized.

      Response: We sincerely thank the reviewer for this important observation. We have now carefully revised the entire manuscript and corrected all scientific names to be properly italicized according to microbiological nomenclature conventions.

2. Authors are requested to provide more information about primer design, specifically, alignments demonstrating absence of mismatches under primers and the number of aligned genomes. The same can be said about alignment with of different biovars, because this is crucial for specificity of NAATs.

      Response: We sincerely appreciate this valuable feedback. In response to the reviewer's comment, the requested information has been incorporated into Section 2.4.1 of the revised manuscript.

3. Rpm is used instead of rcf.

Response: Thank you for pointing out this technical detail. We have converted all centrifugation parameters from rpm to relative centrifugal force (×g) throughout the manuscript.

4. 3.2. Traditional PCR amplification and 2.4.2. Optimization of reaction conditions — important details are missing including reaction volume and reagents concentrations.

      Response: We have now provided complete reaction details as suggested.

5. Page 4, line 157: “in section 1.3.3.” — it seems that the actual number is different from the given one.

      Response: We have corrected all section references throughout the manuscript.

6. 4.4. Specific test — what were DNA concentrations?

      Response: upplement under subsection 2.2.4 : all bacterial genomes were diluted to a concentration of 1ng/μL.

7. 4.5. Sensitivity test — how many technical repeats were analyzed and how was the LoD calculated?

      Response: Add to this section: Under Following optimization of qPCR reaction parameters, triplicate experimental replicates were performed to determine the minimum reliably detectable copy number (The coefficient of variation (CV) for CT values was determined to be less than 1%, which was designated as the limit of detection (LOD).

8. 4.7. Clinical sample testing — a brief description of the purification protocol is necessary. Response:Add to this section: After centrifugation at 4000 rpm for 20 minutes, viral genomic RNA was extracted from the supernatant using a viral DNA/RNA extraction kit. Genomic nucleic acid was removed prior to reverse transcription into cDNA for subsequent analysis.” — why were gDNA discarded if it was actually targeted by the designed assay?

      Response: We thank the reviewer for this important suggestion. We have now provided a detailed description of the complete purification protocol, including both bacterial enrichment from nasal swabs and DNA extraction steps and the identified error has been rectified accordingly. We hereby solemnly declare: The experiment utilized bacterial DNA, not RNA, with all relevant components being modified synchronously. This error constitutes a mere textual description discrepancy and has no impact on the experimental design or results.

9. Amplification results of target genes — what sample was used for the amplification? Also, Figure 1 does not represent the PCR results as it mentioned in the text.

      Response: The bacterial strain employed in this study was Corynebacterium pseudotuberculosis(C. pseudotuberculosis) biovar ovis FJ-PN, as detailed in Section 2.1 of this manuscript. We have corrected the image numbering error.

10. Optimization of SYBR Green I qPCR reaction system — the results of optimization experiments would also be interesting, especially, a gradient of annealing temperature.

      Response: We added the following in 3.4 section: The annealing temperature optimization results showed that the amplification efficiency was highest at 56°C (101%),  higher than those at 58°C and 60°C. Melting curve analysis indicated that the Tm value of the product at 56°C was (81.69±0.22)°C, with a single peak, indicating good amplification specificity. In contrast, a non-single peak peak appeared at 52°C and 54°C, suggesting non - specific amplification.

11. Clinical specimen validation — what were Cq values of discordant samples? Also, the analysis would be more robust, if the samples were tested by an independent previously reported and commonly using method.

      Response: Upon re-examining our methodology, we confirm that we using Cloning and sequencing of amplified products, a commonly used method. The sequencing results showed that the amplified products were consistent with C. pseudotuberculosis biovar ovis, which supports the reliability of our results. We apologize for not emphasizing this earlier in the manuscript—we will add this detail in the revised version.

12. Authors are requested to compare characteristics of their test with previously published and discuss possible limitations of this approach. Also, it is not directly stated why an intercalating dye was used instead of TaqMan or other similar probes. The latter approach is more specific and less prone to false-positive results due to non-specific amplification of primer-dimes and other non-targeted DNA sequences.

      Response: In comparison to previously reported conventional PCR methodologies, the SYBR Green I quantitative PCR (qPCR) assay established in this study exhibits significantly enhanced detection efficiency. Conventional PCR relies on sequence-specific primer binding to target regions for nucleic acid amplification, with results typically visualized via gel electrophoresis. This approach is characterized by operational complexity, requiring a detection cycle of 4–6 hours, and demonstrates a relatively high limit of detection (10³–10⁴ CFU/mL). Furthermore, the open-tube electrophoresis procedure inherent to conventional PCR is susceptible to aerosol contamination, thereby increasing the risk of false-positive outcomes.

Multiplex fluorescent quantification techniques necessitate multiple primer sets, while TaqMan-based assays require costly fluorescently labeled probes. In contrast, the SYBR Green I-based qPCR method eliminates the requirement for probe design and optimization, streamlines the experimental workflow, and offers greater cost-effectiveness compared to TaqMan assays. Notably, for applications involving multiplex detection or large-scale sample screening—where multiple probes would otherwise be required—the SYBR Green I-based approach presents a more accessible alternative for conventional diagnostic laboratories with constrained budgets.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript describes the establishment of qPCR detection method of C. pseudotuberculosis biovar ovis (Cp-bo) targeting CP40 gene. The flow of research and establishment of method seem to be appropriate and scientifically sound. However, descriptions of each chapter are somewhat lengthy and dispersed. The whole manusript is expected to be more concise, showing minimum information, while supplementary materials may be also used. This reviewer points out followings.

1. Objective of ths study should be more clearly focused and described.  Is it aimed to establish PCR detection of Cp-bo first time? Otherwise, is it to improve previously reported detection method? If so, what point is improved? To increase sensitivity?
2. Authors established highly sensitive qPCR method. However, as a practical issue, how is the result interpreted? If even very low amount of Cp-bo is detected by this method, is the animal regarded as being infected? Is there no colonization for this bacterium? If we judge the occurrence of infection with this bacterium, how much amount of bacterial cells must be detected by thismethod? Such discussion is preferable to be added. 
3. Bacterial species names should be rephrased. C.pseudo,..., S.aureus, M.haemolytica should be C. pseudo..., S. aureus, M. haemolytica.
4. 2.3.2. "Traditional" should be "Conventional".
5. 2.3.3. How did authors determined sequence? Method should be added.
6. 3.1, Results, line 206. although authors wrote "a size of 92bp (Figure 1)", figure 1 shows phylogenetic tree. Text seems to show a wrong figure.
7. Method of phylogenetic anakysis (how to make phylogenetic tree) is not written. It should be added. 
8. Figure 2,3,4: Consider whether to add all these figures appropriate or not. 

Author Response

1. Objective of ths study should be more clearly focused and described.  Is it aimed to establish PCR detection of Cp-bo first time? Otherwise, is it to improve previously reported detection method? If so, what point is improved? To increase sensitivity?

      Response 1: Discussion Addendum:This study establishes the first SYBR Green-based fluorescent quantitative PCR detection method for C. pseudotuberculosis biovarovi, addressing the technical gap in molecular detection of this pathogen. Conventional PCR assays exhibit limited sensitivity (10³–10⁴ CFU/mL) and necessitate post-amplification electrophoresis, introducing risks of aerosol contamination. Existing multiplex qPCR protocols require multiple primer sets, complicating experimental design. The developed SYBR Green I qPCR assay demonstrates enhanced sensitivity (10² CFU/mL), employs a single primer pair, features streamlined operation, and enables detection within 60 minutes, rendering it superior for clinical diagnostics and epidemiological surveillance of C. pseudotuberculosis biovarovi.

2. Authors established highly sensitive qPCR method. However, as a practical issue, how is the result interpreted? If even very low amount of Cp-bo is detected by this method, is the animal regarded as being infected? Is there no colonization for this bacterium? If we judge the occurrence of infection with this bacterium, how much amount of bacterial cells must be detected by thismethod? Such discussion is preferable to be added. 

      Response 2: In response to the reviewers' comments, the following methodological details are supplemented to the Discussion section: The limit of detection (LoD) was defined as a cycle threshold (Ct) value ＜35. Bacterial cultures were standardized to an optical density (OD) of 0.6 prior to 10-fold serial dilution for colony-forming unit (CFU) enumeration. A Ct value ＜35 at a bacterial concentration of 100 CFU/mL indicates stable detection and confirms bacterial colonization. Ct values exceeding 35 should be interpreted with caution, considering potential sample contamination or absence of host colonization. Triplicate technical replicates are required to verify result consistency. Inconsistent amplification suggests sample contamination, while consistent positive results indicate either host colonization or environmental presence of C. pseudotuberculosis biovar ovi, necessitating implementation of isolation protocols or environmental decontamination procedures. Concomitant bacterial isolation and serological assays should be performed to validate fluorescence quantification results, with subsequent implementation of targeted isolation and therapeutic interventions as clinically indicated.

3. Bacterial species names should be rephrased. C.pseudo,..., S.aureus, M.haemolytica should be C. pseudo..., S. aureus, M. haemolytica.

      Response 3: We modified the bacterial names and checked the full text.

4. 3.2. "Traditional" should be "Conventional".

      Response 4: We have corrected this statement.

5. 3.3. How did authors determined sequence? Method should be added.

      Response 5: In response to the reviewer's comment, the requested information has been incorporated into Section 2.4.1 of the revised manuscript.

6. 1, Results, line 206. although authors wrote "a size of 92bp (Figure 1)", figure 1 shows phylogenetic tree. Text seems to show a wrong figure.

      Response 6: Thank you for pointing out the error. We have revised the image numbering and supplemented the electrophoresis images of the amplification sequences.

7. Method of phylogenetic anakysis (how to make phylogenetic tree) is not written. It should be added. 

      Response 7: In Section 2.3.3, we discussed the analysis of evolutionary trees. As this topic may not be immediately apparent, we have dedicated a separate section to it.

8. Figure 2,3,4: Consider whether to add all these figures appropriate or not.

      Response 8: Figure renumbering has been implemented: Original Figures 2-4 have been reassigned as Revised Figures 4-6. Figure 4 presents the standard curve, validating the qPCR assay's quantitative reliability (R²=1.00) with optimal amplification efficiency (101%). Figure 5 illustrates specificity analysis, wherein target-specific melting curve profiles confirm amplicon uniqueness and preclude cross-reactivity with non-target pathogens. Collectively, these figures provide critical validation of assay performance and specificity, with each containing unique, text-irreplicable data. Figure 6 (sensitivity curve) is designated as optional and will be omitted if constrained by journal formatting requirements, with further adjustments to be implemented per editorial guidance.

Reviewer 3 Report

Comments and Suggestions for Authors

In the study “Establishment and Application of a SYBR Green I qPCR 2 Detection Method Based on the CP40 Gene of Corynebacterium 3 pseudotuberculosis biovar ovi”, researchers focused on the CP40 gene, a key virulence factor of C. pseudotuberculosis, and developed a rapid and accurate detection method. Genetic analysis revealed that the CP40 gene is highly conserved among C. pseudotuberculosis biovar ovis strains, while showing low similarity to other Corynebacterium species, making it an excellent molecular marker for specific identification.

To enable efficient diagnosis, the authors designed species-specific primers and optimized a SYBR Green I-qPCR assay. The method demonstrated high sensitivity (detecting as few as 52 copies/μL), strong specificity, and excellent reproducibility.

The authors conclude that CP40 not only plays a critical role in bacterial virulence—thanks to its glycosidase activity that helps evade host immune defenses—but also provides a reliable target for diagnostic purposes. The developed qPCR assay combines visualization capability, rapid turnaround, simplicity, and low cost, making it a powerful tool for early detection and integrated control of CLA. This approach could significantly contribute to reducing the global spread of the disease.

In my opinion, the manuscript is well-written, highly readable, and logically organized, making it easy to follow.

Despite this, I have some questions and suggestions:

Materials and methods

Line 195, section 2.4.7. The authors mention extracting viral RNA for testing clinical samples. For bacterial identification, DNA extraction is typically performed, as indicated in section 2.3.2. (line 130). Please clarify the rationale for RNA extraction in this context.

Conclusions

The conclusions correctly highlight the relevance of the CP40 gene as a specific molecular marker to identify Corynebacterium pseudotuberculosis biovar ovi and emphasize the value of the SYBR Green I-based qPCR technology for rapid and accurate disease diagnosis. Nonetheless, in my opinion, a few things could be done better:

-Practical applicability: Although the method is said to be quick, sensitive, and specific, a more thorough explanation of how it might be used in actual situations, like resource-constrained farms or rural areas, would be beneficial.

-Comparison with other diagnostic techniques: A more thorough comparison with current diagnostic techniques, emphasizing the benefits and drawbacks of the suggested approach, would improve the conclusions.

-Future perspectives: Including future perspectives, such as the potential to develop similar tests for other C. pseudotuberculosis biovars or other infectious diseases, would add value.

References

Bibliographic entries in the text and bibliographic entries in the references section do not match correctly due to a shift in the bibliography (see References number 13). Please, correct.

Author Response

1. Line 195, section 2.4.7. The authors mention extracting viral RNA for testing clinical samples. For bacterial identification, DNA extraction is typically performed, as indicated in section 2.3.2. (line 130). Please clarify the rationale for RNA extraction in this context.

      Response1: We sincerely regret the erroneous description of the extraction procedure as RNA extraction. This correspondence formally confirms that all experimental procedures detailed in this section were performed in strict adherence to standardized bacterial DNA extraction protocols, with all generated data and subsequent conclusions derived exclusively from DNA samples. The discrepancy is limited to textual representation and has not compromised the integrity of the experimental design, data analysis, or result interpretation.

The relevant section has been revised to accurately reflect the DNA extraction protocol (refer to revised manuscript). We extend our sincere apologies to the editorial board and reviewers for any inconvenience incurred and affirm our commitment to enhancing the rigor of manuscript preparation in future submissions.

2. Practical applicability: Although the method is said to be quick, sensitive, and specific, a more thorough explanation of how it might be used in actual situations, like resource-constrained farms or rural areas, would be beneficial.

      Response 2: We appreciate the recommendations from the review experts and have incorporated the following additions to the discussion section: The SYBR Green I qPCR detection method established in this study demonstrates significant application potential in resource-limited settings. Although the initial investment in real-time fluorescent quantitative PCR (qPCR) instruments is relatively high, this method offers distinct advantages in terms of operational costs, detection throughput, and timeliness. For primary care settings, it is recommended that veterinarians collect nasal swab samples and transport them to regional testing centers for centralized analysis using foam boxes with ice packs to maintain low temperatures.

The advantages of this model include: Sample stability: Corynebacterium pseudotuberculosis exhibits strong environmental resistance and can survive for several months at low temperatures. Detection efficiency: A single run can process 96 samples, making it suitable for population screening. Cost-effectiveness: The reagent cost per sample is lower than that of probe-based methods or bacterial isolation and identification. Rapid turnaround: Results can be obtained within hours from sample receipt, demonstrating significantly higher efficiency compared to methods such as bacterial isolation and identification or ELISA. For CLA prevention and control, this method enables early identification of infected animals, facilitating timely isolation and management, thereby effectively interrupting the transmission chain of pathogens within livestock populations.

3. Comparison with other diagnostic techniques: A more thorough comparison with current diagnostic techniques, emphasizing the benefits and drawbacks of the suggested approach, would improve the conclusions.

      Response 3: There is currently no report on the SYBR Green I fluorescence quantitative PCR method for C. pseudotuberculosis, and no established method for diagnosing C. pseudotuberculosis biovar ovi. This study establishes the first SYBR Green-based qPCR detection method for C. pseudotuberculosis biovar ovi, addressing the technical gap in molecular detection of this pathogen. Conventional PCR assays exhibit limited sensitivity (10³–10⁴ CFU/mL) and necessitate post-amplification electrophoresis, introducing risks of aerosol contamination. Existing multiplex qPCR protocols require multiple primer sets, complicating experimental design. The developed SYBR Green I qPCR assay demonstrates enhanced sensitivity (10² CFU/mL), employs a single primer pair, features streamlined operation, and enables detection within 60 minutes, rendering it superior for clinical diagnostics and epidemiological surveillance of C. pseudotuberculosis biovar ovi. 

4. Future perspectives: Including future perspectives, such as the potential to develop similar tests for other pseudotuberculosis biovars or other infectious diseases, would add value.

      Response 4: Thank you for your suggestion, We added the following content at the end:The SYBR Green I qPCR platform based on the CP40gene is highly expandable. Future directions:

1.Expand detection scope​

Develop a specific assay for C. pseudotuberculosis biovar equi(broader host range).

2.Optimize technology​

Develop lyophilized reagents requiring only template addition.

5. References

Bibliographic entries in the text and bibliographic entries in the references section do not match correctly due to a shift in the bibliography (see References number 13). Please, correct.

     Response 5: Thank you for pointing out the error. We have noticed it and will make the necessary changes.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Many thanks to authors for their thoughtful replies and careful corrections of the manuscript which quality was significantly improved. The most questions from the first review were properly addressed and do not need further clarifications. However, several issues should still require more attentions. Please find them below.

1. It is recommended to analyze more than 3 technical replicates of samples with a template concentration close to an assumed LoD—16, 32, or more—for a robust estimation of LoD due to relatively unstable and stochastic amplification if the template concertation is low.
2. Direct mentioning of the template type in the section 3.1 will increase readability and prevent possible confusion because authors used several types of samples to validate their PCR test.
3. The question about validation by an independent method assumed usage of an independent analysis, for instance, PCR test reported by another group. Sequencing is a solid option, but it cannot rule out false-positive results caused by sample cross-contamination or contamination by PCR products from previous runs. Also, possibility of false-negative testing should also be considered, and sequencing of PCR products cannot be applied to clarify these issues.
4. Authors are encouraged to discuss limitations of intercalating dyes for detection of qPCR results meaning possible false-positive results due to non-specific amplification and the need to careful melting analysis of amplification products.

Author Response

Comment 1. It is recommended to analyze more than 3 technical replicates of samples with a template concentration close to an assumed LoD—16, 32, or more—for a robust estimation of LoD due to relatively unstable and stochastic amplification if the template concertation is low.

Response：Thank you for the reviewer's professional comments! To further verify the robustness of the Limit of Detection (LOD), we supplemented experiments with low-concentration templates close to the original LOD (52 copies/ μL). The detailed results and analysis are as follows:

1. Supplementary Experimental Design and Results

After thawing the standard plasmid (PMD19T-CP40), we prepared two low-concentration samples via gradient dilution: 26 copies/ μL and 10.4 copies/ μL . Each concentration was subjected to 5 technical replicates of SYBR Green I qPCR under the optimized reaction conditions . The results are presented in the table below:

Concentration(copies/ μL)	Ct Values					CV values
	1	2	3	4	5
10.4	37.52	38.15	-	-	36.95	1.6%
26	35.76	36.33	36.81	36.62	35.95	1.21%

Note: "-" indicates no valid amplification curve.

2. Result Analysis and Basis for LOD Determination

2.1 Amplification Characteristics of Low-Concentration Templates

For the 26 copies/ μL group: The mean Ct value was 36.29 ± 0.44, which was close to the theoretical Y-intercept of the standard curve (36.04) with qualified specificity confirmed by melting curves. However, the CV value was 1.21%, exceeding the reproducibility standard set in this study (CV < 1.0%), indicating insufficient amplification stability at this concentration.

For the 10.4 copies/ μL group: Not only was the CV value as high as 1.6%, but 2 out of 5 replicates showed no amplification. The significant increase in amplification randomness failed to meet the definition of LOD as "stable detection," so this concentration was excluded from consideration.

2.2 Core Reasons for Selecting 52 copies/ μL as LOD

Reproducibility and Reliability: The Ct value corresponding to this concentration was 34.89 ± 0.2, with an intra-assay CV of only 0.48% (original experimental data). It meets the core methodological requirements for LOD ("stable detection with low CV values") and avoids result deviations caused by amplification randomness of low-concentration templates.

Avoiding Inherent False Positive Risks of SYBR Green I qPCR: SYBR Green I dye binds to all double-stranded DNA. Even with highly specific primers, non-target fragments may be amplified after more than 35 cycles—especially in complex clinical samples that may contain species with gene sequences highly homologous to Corynebacterium pseudotuberculosis biovar ovis. Most existing dye-based qPCR studies and commercial kits adopt Ct value ≤ 35 as a reliable positive threshold. Amplification results with Ct value > 35 require cautious interpretation due to increased false positive risks, which is consistent with the requirements for "reliability of low-concentration template results" in the MIQE guidelines.

Practical Significance for Clinical Application: 52 copies/ μL and 26 copies/ μL are highly close in magnitude, corresponding to minimal differences in bacterial load in clinical samples (both at the 10² CFU/mL level), this difference will not lead to significant missed detections. Meanwhile, the strict positive determination standard can reduce false positives, making it more suitable for the clinical needs of "early screening + accurate isolation" in CLA prevention and control.

3. Conclusion

Based on the supplementary experimental results, inherent limitations of SYBR Green I qPCR, and clinical application scenarios, we confirm that 52 copies/ μL is a more rigorous and reliable LOD value. This value not only meets the methodological requirements of "stable detection" but also effectively avoids false positive risks associated with the amplification of low-concentration templates. It does not sacrifice the reproducibility and clinical practicality of results for the sake of pursuing an low numerical value, which is more in line with the core goal of this study to "provide technical support for CLA surveillance and control."

 

Comment 2. Direct mentioning of the template type in the section 3.1 will increase readability and prevent possible confusion because authors used several types of samples to validate their PCR test.

Response: Thank you for pointing out the issue. The section has been revised to the template type should be specified as 'genomic DNA of Corynebacterium pseudotuberculosis biovar ovis FJ-PN strain'.

 

Comment 3. The question about validation by an independent method assumed usage of an independent analysis, for instance, PCR test reported by another group. Sequencing is a solid option, but it cannot rule out false-positive results caused by sample cross-contamination or contamination by PCR products from previous runs. Also, possibility of false-negative testing should also be considered, and sequencing of PCR products cannot be applied to clarify these issues.

response: We have referred to the article by Spier S J (2004), which provides a multiplex fluorescent quantitative method for detecting Corynebacterium pseudotuberculosis based on the taqman-qPCR. Using this method, two samples(Negative in conventional PCR but positive in qPCR) yielded positive results, and these results have been added to this paper.

 

Comment 4. Authors are encouraged to discuss limitations of intercalating dyes for detection of qPCR results meaning possible false-positive results due to non-specific amplification and the need to careful melting analysis of amplification products.

Response: Thank you for the comment regarding the limitations of intercalating dyes in qPCR detection. We have supplemented and refined the relevant discussion in the manuscript to clarify the potential false positive risks of SYBR Green I and the corresponding mitigation measures, as follows: 

Despite these strengths, the methodology relies on SYBR Green I-based qPCR, which introduces limitations due to the dye’s properties: as an intercalating dye, SYBR Green I lacks sequence specificity, it binds all double-stranded DNA, risking false-positive signals (e.g., primer dimers, non-specific bands) during amplification [31]. To address this limitation, a two-tiered optimization framework was implemented as follows: first of all, preventive optimization: Primer sequences were computationally optimised using Primer Premier 5.0 (dimer formation risk prediction and minimization) and BLAST (cross-species non-specific binding validation; Fig. 1). Annealing temperature gradient screening established 56 ℃ as the optimal reaction condition, which effectively suppressed primer dimer formation and yielded a single melting peak (Tm = 85.69 ± 0.22 ℃; Fig. 4B). Results were validated through combined analysis of amplification curve Ct values and melting curve profiles. Secondly, Confirmatory validation: Suspected positive samples were subjected to TaqMan-qPCR (accuracy enhancement via probe-based specificity) and gel electrophoresis/cloning-sequencing. These sequential steps ensured robust control of non-specific amplification artifacts.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version is well improved.

Author Response

We sincerely appreciate your positive evaluation and recognition of our manuscript, as well as your valuable suggestion for its acceptance. Your affirmation is a great encouragement to us. We have carefully checked and polished the full text of the manuscript to further ensure the accuracy of data expression and the fluency of academic writing. We will strictly follow the journal’s format requirements for the final revision and submission. Thank you again for your professional and constructive comments, which have provided important support for the smooth progress of our manuscript submission.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have provided clear and exhaustive responses to all comments and requests. In my view, the manuscript is suitable for acceptance in its present form.

Author Response

We sincerely appreciate your positive evaluation and recognition of our manuscript, as well as your recommendation for acceptance. Your professional affirmation is of great significance to us. We have further polished the full text for language accuracy and format standardization in accordance with the journal’s requirements to ensure the quality of the manuscript. Thank you again for your valuable support for this submission.