PONE-D-23-14092A novel rapid detection method for a single-nucleotide substitution mutation derived from canine urothelial and prostatic carcinoma cells present in small amounts in urine sedimentsPLOS ONE

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2.  In your manuscript, you appear to indicate that urine sediments of dogs suspected of having urothelial and prostatic carcinoma were obtained and used in your study. However, there is no description of where the urine samples were obtained and permission. Please clarify. 

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

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

Reviewer #1:

The authors present interesting work aimed at developing an inexpensive method to detect the BRAFV595E mutation in the urine of dogs. They point out the shortcomings of other assays and the expense of other assays.

The authors clearly describe their new approach, the experiments performed, and the results. Their strategy for the assay is intriguing.

The finding that there were samples that were negative for the mutation by Sanger sequencing and positive for the mutation with the new assay were not surprising, and the authors presented followup work to explain the enhanced sensitivity of their new assay. Defining the specific percentage of alleles with the mutation which are required for a positive test by either method, however, requires further study.

One of the main concerns about the research is that the results were not compared to ddPCR, which is the most widely available commercial assay to detect the BRAF mutations in dog urine. Although the ddPCR test is expensive through commercial labs, it can actually be performed for a fraction of that cost. The commercial labs overcharge for the assay. Based on the sentence starting on line 385, is the reader to conclude that the new assay is more expensive than the ddPCR assay?

On line 134, please explain how the samples were randomly selected? How many samples were available? How ere the 22 samples selected?

Could the authors explain the sentence starting on line 297 more clearly. In what instances did a veterinarian require discrimination of the gene mutation? Was it for targeted therapy, or was it related to a diagnosis? It is becoming more obvious that the presence of the mutation does not always correlate with the presence of clinical cancer. This is being recognized by multiple veterinary specialty centers, and the poor predictive value was confirmed by a study published in 2022. https://pubmed.ncbi.nlm.nih.gov/36439482/

The sentence starting on line 43 is not necessarily correct. The percentage of cells in a tumor mass that harbor the BRAFV595E mutation is thought to vary considerably from tumor to tumor. And the ratio of tumor cells to normal urothelial cells in the urine also varies from dog to dog. If there are referenced studies to back up this sentence, please add those references. Otherwise, it would be best to remove this sentence.

This same comment applies to the sentences starting on line 377.

Can the authors please clarify the sentence starting on line 384. Did the third-generation sequencing match with the results of the new assay for the 3 samples that were positive by the new assay and negative via Sanger sequencing? If the authors are confident in that result, then it’s not necessarily a “false positive”, rather it shows higher sensitivity with the new assay. It would be best to simply state that there were 3 samples in which the mutation was detected by the new method, and not by Sanger sequencing.

Reviewer #2: 

The author has successfully developed a straightforward, rapid, and highly efficient technique for the detection of single nucleotide mutation, exhibiting high sensitivity and economic feasibility. Notably, this method investigates a superiorly stable DNA blocker in comparison to the ORNi-PCR. The experimental design exhibits strong logic and coherence, while the validation of the method remains robust and satisfactory. However, I would like to propose two recommendations that might further refine this paper.

1. Figure 3 delineates data obtained from examining three primer combinations and two blocker concentrations. To better augment the legibility and understanding of this figure, would the author consider incorporating these specific annotations directly into the figure? Such an addition would simplify the interpretation of the data for the readers. Furthermore, it would be beneficial if the meaning of each dot (presumably representing an individual test) could be explicitly mentioned within the figure illustration.

2. In the section entitled "Sensitivity Test", has the author considered to sequencing the resultant products? This could potentially provide an unambiguous manner to demonstrate the efficiencies of the three primer combinations.

I look forward to hearing your thoughts on these suggestions and I appreciate your time and consideration in addressing these recommendations.

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Reviewer #1: No

Reviewer #2: Yes: Yin Tang

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A novel rapid detection method for a single-nucleotide substitution mutation derived from canine urothelial and prostatic carcinoma cells present in small amounts in urine sediments

PONE-D-23-14092R1

Dear Dr. Okumura,

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.

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Kind regards,

Ruslan Kalendar

Academic Editor

PLOS ONE