Abundance of Yersinia pestis Nucleic Acids in Soil from Rattus tanezumi Plague Foci in Yunnan Using ddPCR

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have demonstrated the various presence of Y.pestis nucleic acids in different soil samples, including those from locations with a history of plague incidence.

Major criticism

This is too strong and insufficiently proven to interpret the results as that soil type is a key environmental factor influencing the nucleic acid abundance, or as "this study revealed that the abundance of Y. pestis nucleic acids in the environment is regulated by soil type". 

As the authors noted in 4.5 (LL500-2), 87 samples constitute a small set for nine soil types. The representation of certain soil types was very limited, with other samples being sourced from several locations (Appendix I).  

It is evident that it is premature to draw definitive conclusions regarding the potential regulatory role of soil properties on NA abundance due to the absence of a proposed mechanism that could account for this relationship. Consequently, the primary outcome of this study is the demonstration of variations in the presence of Y. pestis nucleic acids in soil samples from different locations, with no further conclusions on the role of soil types. The results of the statistical treatments provide only a preliminary indication of the potential for the preservation of intra- and extracellular Y. pestis DNA in specific soils or soil groups.

The presentation of results is reminiscent of a technical report. The results of the statistical treatments that indicate no significant differences are extensively rendered in the text and should be moved as supplementary material.

The discussion is overly lengthy. It should be shortened by avoiding repetitions of results and excessive and general information on soil conditions to be essential for DNA preservation.

L13-4: It is incorrect to assert that "this study employed a duplex droplet digital PCR (ddPCR) assay targeting the CAF1 and YPO039 genes to detect 78 soil samples", as this procedure is not capable of detecting soil samples.

L70: It is strongly recommended that more information be provided on the challenges of PCR assays.  

LL82-3: It is mandatory to support that the Caf1 and YPO0392 genes are specific to Y.pestis by providing the relevant reference(s).

Please ensure that references to ddPCR assay and probes (LL133-137) are included.

It is common practice for authors to provide one list of references, not two.

It is generally considered good practice to avoid citing materials from theses and symposia.

A number of the cited works are written in Chinese and are not easy to open via links indicated.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors The topic is important. Understanding environmental persistence of Y. pestis is a long-standing and controversial issue in plague ecology. The use of ddPCR for soil-based quantification is technically appropriate and potentially valuable.   However, major conceptual and methodological issues must be addressed before publication, particularly: •Over-interpretation of eDNA as evidence of ecological reservoirs •Lack of assay validation and inhibition control details •Extremely low copy numbers with questionable biological meaning •Statistical overemphasis relative to biological signal strength •Repeated grammatical and stylistic issues throughout     Scientific rationale and ecological interpretation   Strengths •Addresses a key unresolved question: Does Y. pestis persist in soil? •Uses dual targets (plasmid + chromosomal gene), which is methodologically sound. •Attempts to link soil type with pathogen persistence.   But there are some Major conceptual concerns   line 101. Why was the sampling depth set at 30cm? Y. pestis is often associated with rodent burrows, which can be deeper. A brief justification for this depth would be helpful.   1. Detection of DNA ≠ Evidence of Reservoir   The manuscript repeatedly suggests that certain soils may function as “environmental reservoirs” (Discussion section 4.3, pp. 11–12  ).   This is not justified by the data.   ddPCR detects DNA. It does not demonstrate: •Viable bacteria •Replication in soil •Maintenance between epizootics •Infectious potential   The median FAM copy number is 0.0600 copies and VIC median is 0.0000 (Results 3.1, p. 4  ).   These are extremely low signals, very close to background detection limits.   My recommendation:   Replace all references to: •“environmental reservoir” •“saprophytic phase evidence” •“long-term persistence”   With:   “differential environmental retention or stability of Y. pestis DNA”   Unless viability experiments (culture, PMA-ddPCR, RNA detection) are added.   2. ddPCR Methodology Evaluation   Strengths •Duplex detection (caf1 plasmid + chromosomal gene) •Use of QX200 system (industry standard) •Non-parametric statistics appropriate for non-normal distributions   Major technical concerns

The duplex ddPCR method is a strength, but the description needs more detail. The authors use the caf1 gene (on pMT1 plasmid) and ypo0392 (chromosomal). A key point to discuss is the possibility of plasmid loss in the environment. Detection of the plasmid gene alone could be from cells that have lost the chromosome, or vice-versa. 

The master mix is listed as "Cy5.5". This is likely a typo from a generic kit protocol. For a duplex reaction using FAM and VIC probes, the master mix should be compatible with those fluorophores (e.g., ddPCR Supermix for Probes (No dUTP)). Please clarify.

Positive Determination Criteria: "the copy number... greater than 0." This is a very sensitive criterion. In ddPCR, there can be "rain" events or very low-level background. It's more robust to define a positive based on a statistical threshold, such as the number of positive droplets being above the limit of detection (LOD) derived from negative controls. The authors should state if any no-template controls were run and how they performed.

So, about this critical point: a) Not Limit of Detection (LOD) reported   There is no information on: •Analytical sensitivity •Limit of blank •False positive rate •Replicate variability   Without LOD validation, detection of 0.06 copies may represent stochastic droplets or background.   b) Positive definition is scientifically weak   Page 4:   “A sample was determined as positive if the copy number in either the FAM channel or the VIC channel was greater than 0.”     This is problematic.   In ddPCR, a threshold of >0 copies is insufficient because: •Rain (ambiguous droplets) can occur •Low-level contamination may generate 1–2 positive droplets •Background fluorescence can cause false positives   Recommended revision: •Define minimum droplet count threshold (e.g., ≥3 positive droplets) •Require concordance of both targets OR justify single-target positivity •Include no-template controls and extraction blanks in results   3. Soil PCR inhibition not addressed   Soil is highly inhibitory.   No mention of: •Internal amplification control •Dilution test for inhibition •Spike recovery experiment   This is critical in environmental PCR studies.   4. Extremely Low Copy Numbers   Median values: •FAM median: 0.0600 •VIC median: 0.0000     These values are near the Poisson noise range.   Biologically, what does 0.06 copies mean? •Copies per reaction? •Copies per µL? •Copies per gram of soil?   Units are unclear.   Absolute quantification must be standardized per gram of soil.   4. Statistical Interpretation   Soil Type Effect   Statistically significant difference in copy number among soil types (p < 0.001; Table 3, p. 7  ).   However: •Some soil types have n = 3–5 samples •Outliers (JH-33, YJ-22) heavily influence distribution •Biological significance unclear due to extremely low absolute copy numbers   Statistical significance does not equal ecological significance.     5. “Abundance-distribution decoupling”   The manuscript heavily emphasizes this concept.   This term is not standard in infectious disease ecology literature and may overstate findings.   It would be better to state:   Soil type influenced quantitative signal intensity but not detection probability.

In any case, this is an interesting concept the authors introduce. However, the argument would be stronger if supported by some soil physicochemical data (pH, organic matter, iron content, etc.) to explain why certain soil types (Laterite, Dry Red, Purple) act as "reservoirs" and others (Yellow Soils) act as "sinks." The current discussion speculates on this but lacks data. While the study's scope may not have included this, it should be mentioned as a key future direction.

  6. Discussion: Overinterpretation issues   The manuscript proposes: •Soil-driven ecological filtering •Source-sink dynamics •Environmental reservoirs •Support for saprophytic phase hypothesis   These are speculative and not supported by viability data.   Especially problematic: •Linking iron-rich laterites to persistence without measuring iron •Linking soil microbiome interactions without microbiome data   Recommendation: Reduce speculative language by ~40–50%.  

Section 4.1: The reasoning linking climatic conditions to the high positive rate is logical. The point about the optimal growth temperature of Y. pestis (28-30°C) is slightly off, as this is optimal for in vitro culture. In the environment, it's more about survival, not growth. This nuance could be adjusted.

  7. Figures and Tables   Figure 1. Line 202. Change Fig. 1, Fig. 2 Fig. 3 Fig. 4 in Figure 1. Create a title that includes all the figures and then indicate them with a), b), c) and d). The same for Fig. 5 and 6 and Fig. 7 and 8.   Figures (pp. 5–8  ): •Axis units missing (copies per what?) •No log scale despite highly skewed distribution •Very dense labeling   Recommend: •Log10 transformation •Clear units (copies per reaction or per gram soil) •Indicate LOD line on plots   8. Required Major Revisions   Before publication, authors must: 1.Provide assay validation data (LOD, specificity, blank controls) 2.Define positivity threshold more rigorously 3.Standardize copy number reporting per gram soil 4.Reduce ecological overinterpretation 5.Clarify units and droplet thresholds 6.Substantially revise English 7.Discuss limitation that DNA ≠ viable bacteria

  Comments on the Quality of English Language The manuscript requires major English editing. Below are systematic issues.   A. Consistent capitalization errors   Incorrect: •ddpcr •droplet digital pcr •rouped boxplots •Laterite, Purple soils (inconsistent)   Correct: •ddPCR •Droplet Digital PCR •Grouped boxplots •Laterite soils / purple soils     B. Grammar corrections (only some Examples)   Original:   “To clarify the persistence mechanism of Yersinia pestis in foci…”     Better:   “To clarify the mechanisms underlying environmental persistence of Yersinia pestis…”     Original:   “The results showed that the overall positive rate…”     Better:   “Overall, 65.38% (51/78) of soil samples tested positive for at least one target gene.”     Original:   “Testing for the Abundance of Y. pestis Nucleic Acids…”   Avoid repetitive capitalized section titles. Use sentence case.     Incorrect spacing examples: •“P = 0.0362 .05” •“Epidemic Occurred ” (extra space) •“Appendixs” (should be “Appendices”)   C. Repetitiveness   The phrase:   “The results showed that…”   appears excessively.   Vary sentence structure.     D. Abstract revision recommendation

The objectives are clear, but the language is very repetitive ("Yersinia pestis nucleic acids"). Vary the phrasing (e.g., "pathogen DNA," "target sequences").

Currently overstates ecological implications.

The conclusion is too long. It should succinctly state the main finding: soil type, not historical outbreaks, is the key correlate of Y. pestis eDNA abundance, with implications for environmental monitoring.

Suggested closing sentence revision:   Instead of:   “Soil type is a key environmental factor influencing the nucleic acid abundance…”   Use:   “Soil type was associated with differences in detected Y. pestis DNA copy numbers, although the biological significance of these low-level signals requires further investigation.”

3.31. Descriptive Statistical Analysis: this must be 3.3.1.

Figure 7. rouped boxplots of different Line 296: Figure 7. Grouped boxplots of different

P>0.05 line 30:     P (check all P must be in Italics)

P = 0.0362 .05. line 309 This is not correct

(P = 0.0307 0.05), line 319 This is not correct 

It can be concluded - Line 362: (missing space before It). "It can be concluded..." is a very strong statement from a single correlation. "This suggests that..." is more appropriate.

"Laterites, dry red soils" should be lowercase as they are soil types, not proper nouns: "laterites, dry red soils". Check all manuscript.

6. Patents line 528 Delete

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No further comments

Author Response

Thank you very much for reviewing our manuscript again.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for providing the revised manuscript and the supplementary data. The authors have done an excellent job addressing the major concerns from the previous round of review. The manuscript is now substantially improved: the introduction is complete, the methods are clearer, the statistical analysis is rigorous, and the discussion is balanced and insightful. The use of a defined limit of detection (LoD) rather than a simple “copy number > 0” is a significant improvement that adds credibility to the findings.

I have only a few remaining minor issues and suggestions. After these are corrected, the manuscript will be suitable for publication.

Minor revisions required

1. Abstract

• Line 23: “samples for both caf1 and ypo0392 or ypo0392 positive were considered Y.pestis positive” – This phrasing is ambiguous. Better: “A sample was considered positive if the copy number of ypo0392 exceeded the LoD (regardless of caf1), OR if both genes exceeded their respective LoDs.”
• Line 24:“No correlation was observed between either the positive rate or the copy number of Y. pestis and the classification of an area as a plague focus or non‑focus.” – This is correct for the positive rate (Table 2, p=0.758), but for copy number the Mann‑Whitney test gave p=0.603 (caf1) and p=0.372 (ypo0392), which also show no correlation. The sentence is accurate but consider splitting into two sentences for clarity.

2. Materials and Methods

• Section 2.2 (line 140): The LoD values (8.9 and 15.4 copies/reaction) are given, but the manuscript does not state whether these were experimentally determined in this study or taken from the literature. In the limitations (page 10, lines 418–426), the authors acknowledge that the LoD was referenced from the literature because they did not perform their own limit‑of‑detection experiments. This is acceptablebut the Methods section should explicitly state: “The LoD values used (8.9 copies/reaction for caf1 and 15.4 for ypo0392) were derived from a previous study using the same instrument and similar targets [Zhao et al., 2024].” Currently it is implied but not stated. Please add a brief sentence.

3. Results

• Table 1: The table is well‑organized. However, the current layout is hard to read. Especially the header line. Try simplifying it by using abbreviations and adding a note
• Line 219: “The median copy number of caf1 was generally close to or higher than that of ypo0392.” – This is a descriptive observation. Consider adding a brief explanation in the discussion (already mentioned in section 4.2 that plasmid genes may be more abundant).

4. Discussion

• Section 4.1, line 297: The sentence “Y. pestis nucleic acids had a long persistence capacity in the environment and could be maintained for months or even thousands of years...” – The reference to “thousands of years” (ancient DNA from dental pulp) is correct, but the phrase “in the environment” might be misleading because ancient DNA comes from protected environments (e.g., within teeth). It is fine, but consider adding “under certain conditions” to avoid overgeneralization.
• Section 4.4, line 391: The sentence “Purple soil showed significant differences in pH, organic matter, and medium/trace element contents, compared with other soil type” – should be “compared with other soil types”. Also, a citation is given [33] but that reference appears to be about microplastics in purple soil. The citation is not directly about comparison of purple soil properties to other soil types. Please check the reference or replace with a more appropriate one (e.g., Ma et al. 2023 might be correct, but the topic seems off). If no better reference exists, you could simply state that purple soils are known to have distinct physicochemical properties (a general fact) without a specific citation.

5. Limitations

• First limitation (LoD from literature): This is an honest acknowledgment. However, to strengthen the manuscript, the authors could add a brief sentence in the Methods or Discussion stating that they verified the LoD by running a dilution series of the positive control in a preliminary experiment, if such data exist. If not, the current wording is acceptable but the manuscript would be stronger if the authors had performed their own LoD determination. Given that this is a revision, I do not require new experiments, but the editors may ask. I recommend adding a short sentence: “Future work should include a formal LoD determination using matrix‑matched controls.”

6. Minor language/typo corrections

• Page 1, line 12: “Yongmei LA andFan Li” – missing space: “and Fan Li”.
• Page 1, line 13: “Correspondence: kg1230@163.com(S.Y.)” – missing space after colon. Also, in the author list there are a lot of email: choose only one. Typically, only correspondence email addresses are listed. Please check the journal’s policy.
• Page 2, line 44:“Plague in Rattus tanezumi plague focus of Yunnan” – missing “the” before “Rattus”
• Page 8, line 310: “dissenting views also Y. pestis was found” – missing verb: “dissenting views also hold that Y. pestis was found” or simply “dissenting views also exist: Y. pestis was found...”.
• Page 10, line 419: “meeted the conditions” – typo: “met the conditions”.
• Page 12, reference 6: The author list is abbreviated as “C, S.; B, D.; Q, D.; ...” – This is incorrect. Should be “Su, C.; Duan, B.; Qin, D.; ...”. Please check the original publication and correct the author names.

Chack all bacterial name: must be in Italics

Author Response

Please see the attachment.

Author Response File: Author Response.pdf