Quantitative PCR Assays for the Strain-Specific Identification and Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus X253

Round 1

Reviewer 1 Report

The work describes a genome-based strain-specific assays for probiotc strain L. rhamnosus X253, include strain differentiation, species-identification and enumeration methods. This study is well defined and written, but the following issues should be addressed:

1. The author found 12 strain-specific SNPs that can be applied to differentiate the L. rhamnosus X253 from other 29 L. rhamnosus reference strains (Table S1). However, I briefly checked and found that the strain L. rhamnosus strain P118 (accession number: CP099396) is quite similar to the X253.

After genome-based comparison, the dDDH and ANI values between this two strains are 100% and 99.96%, and the strain-specific SNPs provided in this manuscript such as holA and alaS are also same. 

2. The specificity of the species-specific qPCR primer should include the species L. chiayiensis and L. zeae, which are closely related to the L. rhamnosus, and belong to the L. casei group.

Author Response

Response to Reviewer 1 Comments

Point 1: The author found 12 strain-specific SNPs that can be applied to differentiate the L. rhamnosus X253 from other 29 L. rhamnosus reference strains (Table S1). However, I briefly checked and found that the strain L. rhamnosus strain P118 (accession number: CP099396) is quite similar to the X253. After genome-based comparison, the dDDH and ANI values between this two strains are 100% and 99.96%, and the strain-specific SNPs provided in this manuscript such as holA and alaS are also same. 

Response: Thanks for the comment. In the Genebank database, L. rhamnosus strain P118 is described as a potential probiotic strain isolated from yoghurt. The genomic data for strain P118 were submitted on 26 June 2022, later than our work was completed. An SNP analysis performed on the genomic sequences of all 31 L. rhamnosus strains, including strain P118, revealed that strain X253 could be differentiated from others with four SNP loci of KEM50 05235, KEM50 06745, xerS, and KEM50 10215. It is expected that single SNP site might not be sufficient for strain identification with the release of more genomic data of L. rhamnosus strains. In that case, multiple SNP loci-based analysis might be used for accurate strain-level identification. We added some sentences in the Discussion section (Lines 291-294 on page 10-11).

Point 2: The specificity of the species-specific qPCR primer should include the species L. chiayiensis and L. zeae, which are closely related to the L. rhamnosus, and belong to the L. casei group.

Response: Thank you for your suggestion. As this article focused on the analysis of differences between strains of L. rhamnosus, related strains of L. chiayiensis and L. zeae species were not included in the manuscript. Comparing the genomic sequences of all three strains of L. chiayiensis and seven strains of L. zeae in the NCBI database with those of L. rhamnosus X253, we found that only the SNP locus on the alaS gene could be used to distinguish the strain X253 from the other ten strains. However, the alaS SNP locus did not effectively distinguish strain X253 from strain P118. Therefore, with more genomes being compared and analyzed, multi-SNP locus-base method will be necessary for the identification of strain X253. We added some sentences in the Discussion section (Lines 291-294 on page 10-11).

Author Response File: Author Response.docx

Reviewer 2 Report

In this study, the authors designed and developed a primer set for detecting and quantifying Lacticaseibacillus rhamnosus X253, which could be applied to probiotics products. A primer set and probe that target the SNP of the holA gene were produced after screening 12 SNP loci among 30 distinct L. rhamnosus. Its specificity, stability, sensitivity, repeatability, and reproducibility were confirmed with the sample and actual products. They proved the accuracy and precision of their PCR assay. However, there was little discussion about SNP screening and misleading context. The authors should revise major comments.

P1L39: Lactobacillus should be changed to Lactiplantibacillus.

P2L71-73: Add references

P3L91-92: There was no information on the instrument analyzing the amount of DNA. Also, generally, bioanalyzer is not precise for measurement of DNA concentration.

P3L94-97: More reasonable reasons or scientific evidence were needed for choosing holA loci. Among 12 SNPs, alaS or xerS also could be a genetic marker. Authors should suggest qPCR results of other SNP positions. Additionally, Table 2's labeling of the primers and probe is unclear, and labeling SNP on the probe is clear for understanding. 

P4L111: All genus names must follow the new taxonomy.

P5L134-135: The terms of repeatability and reproducibility were well described. However, a further explanation was needed for how the RSD value of reproducibility was obtained.

P7L184-190: Symbols in figure 2 represent DNA samples, 10, 5, and 2 ng/µL, separately. Square, which is 5 ng/µL sample, showed a lower LOQ (limit of quantification) value than the triangle, which is 2 ng/µL sample. The authors should explain how LOQ value from the high concentration sample was lower than the value from the low concentration sample.

Author Response

Response to Reviewer 2 Comments

Point 1: P1L39: Lactobacillus should be changed to Lactiplantibacillus.

Response: Thanks for the comment. We have changed “Lactobacillus plantarum and L. acidophilus” with “Lactiplantibacillus plantarum and Lactobacillus acidophilus” in the revised version.

Point 2: P2L71-73: Add references

Response: Thanks for the comment. An article concerning possible probiotic properties of L. rhamnosus X253 is in preparation and has yet to be published.

Point 3: P3L91-92: There was no information on the instrument analyzing the amount of DNA. Also, generally, bioanalyzer is not precise for measurement of DNA concentration.

Response: Thanks for the comment. We have added information about the instruments used for the qualitative and quantitative analysis of DNA samples in the revised version and have also provided the corresponding references (ref. [24], [25]). Please see lines 91-95 on page 3.

Point 4: P3L94-97: More reasonable reasons or scientific evidence were needed for choosing holA loci. Among 12 SNPs, alaS or xerS also could be a genetic marker. Authors should suggest qPCR results of other SNP positions. Additionally, Table 2's labeling of the primers and probe is unclear, and labeling SNP on the probe is clear for understanding. 

Response: Thanks for the comment. In this study, we designed primers and probes for all 12 SNP loci, but only the SNP locus on the holA gene demonstrated the best specificity after amplification. We will optimize the primers and probes for the remaining 11 loci in the future studies. Additionally, we have highlighted the SNP on the probe in red in Table 2 to indicate its location.

Point 5: P4L111: All genus names must follow the new taxonomy.

Response: Thank you for your suggestion. All species names in Table 3 have been updated according to the new taxonomy in the revised manuscript.

Point 6: P5L134-135: The terms of repeatability and reproducibility were well described. However, a further explanation was needed for how the RSD value of reproducibility was obtained.

Response: Thanks for the comment. As you suggested, we added a sentence “RSD values for repeatability and reproducibility are calculated as the mean Cq of the same sample measured at different times and on different platforms, respectively.” in the revised manuscript (Lines 139-141 on page 5).

Point 7: P7L184-190: Symbols in figure 2 represent DNA samples, 10, 5, and 2 ng/µL, separately. Square, which is 5 ng/µL sample, showed a lower LOQ (limit of quantification) value than the triangle, which is 2 ng/µL sample. The authors should explain how LOQ value from the high concentration sample was lower than the value from the low concentration sample.

Response: Thanks for the comment. We apologize for any confusion caused by the lack of clarity in Figure 2. A new line at the Cq value of 35, representing the limit of quantification, has been added in Figure 2, and the points above 35 have been removed to offer a better illustration of limit of quantification. In addition, results of linear regression were also added in the figure. According to the regression equation, the limit of quantification was calculated to be 0.00029, 0.00027 and 0.00022 ng/µL, for Dilution series 1, 2, and 3 respectively, which are essentially the same. 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have responded and revised the manuscript.

Author Response

Point: The authors have responded and revised the manuscript.

Response: Thanks.

Author Response File: Author Response.docx

Reviewer 2 Report

Authors give a clear answer to reviewer’s comments.

Author Response

Point: Authors give a clear answer to reviewer’s comments.

Response: Thanks. 

Author Response File: Author Response.docx