A Quadruplex Reverse Transcription Quantitative Polymerase Chain Reaction for Detecting Canine Coronavirus, Canine Rotavirus, Canine Parvovirus, and Canine Distemper Virus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

 

L24-25. Unrealistic precision. 10² is better.

L45. Popular -> frequent.

L71. ‘uneasy contamination’ - > ‘reduced risk of contamination’ 

L87. First sentence. ‘This study did not involve any experiments on live animals’ would be a better phrasing.

L102-106. Clinical samples. This description is rather brief. What kind of sample were they and in what form were they supplied?

L116-126. What was the procedure for selection of primers? If any form of primer design/selection software was used, this should be stated. If, as appears to be the case, the authors merely searched the multiple sequence alignments for conserved sequences and then adjusted the length of the primers until they got the required Tm, then they should state this. Also, which method was used for determining Tm?

The authors may be interested to know that calculated Tm values may differ quite a lot from the true value: see Pedersen BN, Jenkins A, Paulsen KM, Basset C, Andreassen ÅK. Development of a real-time PCR method for detection of European and Siberian subtype of tick-borne encephalitis virus. Microbiol Res, 2023, 14, 1545–1560. https://doi.org/10.3390/microbiolres14040106

 

The software used for generating multiple sequence alignments should be stated.

Looking at the primer sequences, I notice some design errors. CCoV(M)-F has the self-complementary sequence -AATT at the 3’ end, and CRV(VP79-F has -GC. Both of these are likely to form primer-dimers, which may explain why the analytical sensitivity is not higher. Optimally-designed PCR tests typically achieve single-molecule sensitivity with plasmid controls in the absence of interference from background DNA. In addition, the 3’ -CC of CCoV (M)-R is complementary to the 3’ -GG of CDV(N)-F and CPV(VP2)-P, although in the latter case, depending on the exact placing of the BHQ3, dimer-formation may be blocked by the quencher.

 

Figure 1. Multiple sequence alignments. The font size is very small, and the figure will very likely become unreadable in the final printed version. Since many of the sequences are identical, I suggest removing sequence duplicates from the alignments. The accession numbers of these may be mentioned in the figure text. It will probably be advantageous to make four separate figures. With the reduced number of lines, it should be possible to put each figure in landscape format, which would allow an acceptable font size. Consider also removing the column with the strain name. The accession number is enough.

It would also be an advantage to add a few (3 – 5) sequences from the most closely related virus types; this would allow the reader to evaluate the possibility of cross-reactions. If there is insufficient space, a second series of figures would be appropriate.

L154-164 and L208-216. Plasmid constructs. L208-216 is a much better methodological description and could with advantage replace much of L154-164 in the methods. This information belongs in the methods, rather than the results.

L205-206. Reference PCR. The reference PCR is central to the study and needs to be described. The description should include (a) the analytical sensitivity of the reaction (if known) (b) the target genes (c) how the assay was implemented. The authors say that it is a multiplex reaction, but imply it is not a quadriplex - so how it was multiplexed in order to detect all four virus types is relevant.

L250-254. LODs. Three decimal places is unrealistic precision. Express instead as whole numbers or in scientific notation with two significant figures, e.g. 111.440  ≈ 111 ≈ 1.1x10². Elsewhere (e.g the abstract), these figures can be summarised to ca. 10² copies for all four virus types.

3.7. The range of Ct values observed would be relevant. This would allow the repeatability data to be related to values realistic in clinical samples.

L274-276. Coinfections. This sentence is very difficult to read. It would be better to mention the two most important coinfections and refer to the table for further details.

L277-278. Sensitivity and specificity. It is not clearly stated what specificity and sensitivity are calculated relative to. If it is the reference assay, then this sentence belongs in the next paragraph after the positivity rates for the reference assay.

L279-284. How well did the two assays agree in detection of coinfections? This is a more demanding task and thus a better test of assay quality. As the numbers will be small, simple descriptive statistics would be sufficient. It would also be interesting to know if the discrepant samples were concentrated among the coinfections.

L283 and elsewhere. ‘reported reference assay’. Just ‘reference assay’ would be better.

L289-297; 298-303. This information is in the tables and does not need to be repeated. Table text should be reserved for notes to explain the text, e.g. abbreviations and symbols.

Table 6, Table 7 and elsewhere. ‘The established multiplex RT-qPCR’; The reference multiplex RT-qPCR’. These are rather cumbersome terms. Why not ‘(the) current PCR’; (the) reference PCR’?

311-319. This belongs in the introduction, where much of what is written has already been said. The section on previously developed tests should also mention other detection methods (serology, cultivation) if they exist. This would be a good place for a general description of the reference PCR. A point which the authors seem to have overlooked is that the reason no quadriplex PCR has previously been developed is that many real time PCR systems are incapable of 4-channel multiplexing. This also places limitations on the implementation of this test in other laboratories, as these instruments are still not standard equipment, and are quite expensive. The authors should mention this, and perhaps suggest ways of implementing their test on simpler instruments.

Repeatability. This would be more easily grasped if expressed as a range of C_t values and should be accompanied by the number of target copies. It occurs to me that the authors’ LOD experiments would afford (intra-assay) repeatability for copy numbers near 10².

Comments on the Quality of English Language

The standard of English is generally satisfactory throughout. 

Author Response

Revision Notes

April 27, 2024

Dear editor,

The manuscript has been revised carefully according to the editor’s suggestions and the reviewers’ suggestions. The details are as follows.

 

Reviewer #1

Comments and Suggestions for Authors

1. L24-25. Unrealistic precision. 10²is better.

Response: The LODs have been changed to “1.1 × 10²”. Please see Line 25 in the revised manuscript.

2. L45. Popular -> frequent.

Response: “popular” has been revised to “frequent”. Please see Line 46 in the revised manuscript.

3. L71. ‘uneasy contamination’ - > ‘reduced risk of contamination’ 

Response: “uneasy contamination” has been revised to “reduced risk of contamination”. Please see Line 72 in the revised manuscript.

4. L87. First sentence. ‘This study did not involve any experiments on live animals’ would be a better phrasing.

Response: The first sentence has been changed to “This study did not involve any experiments on live animals”. Please see Lines 89-90 in the revised manuscript.

5. L102-106. Clinical samples. This description is rather brief. What kind of sample were they and in what form were they supplied?

Response: The clinical samples include feces, anal and nasal swabs from each dog with different signs of gastroenteritis. All samples were placed in a specialized medical ice box, and transported to our laboratory under ≤4 ℃ condition within 12 h after collection. This information has been added in the revised manuscript. Please see Lines 111-120 in the manuscript.

6. L116-126. What was the procedure for selection of primers? If any form of primer design/selection software was used, this should be stated. If, as appears to be the case, the authors merely searched the multiple sequence alignments for conserved sequences and then adjusted the length of the primers until they got the required Tm, then they should state this. Also, which method was used for determining Tm?

The authors may be interested to know that calculated Tm values may differ quite a lot from the true value: see Pedersen BN, Jenkins A, Paulsen KM, Basset C, Andreassen ÅK. Development of a real-time PCR method for detection of European and Siberian subtype of tick-borne encephalitis virus. Microbiol Res, 2023, 14, 1545–1560. https://doi.org/10.3390/microbiolres14040106

 The software used for generating multiple sequence alignments should be stated.

Looking at the primer sequences, I notice some design errors. CCoV(M)-F has the self-complementary sequence -AATT at the 3’ end, and CRV (VP7)-F has -GC. Both of these are likely to form primer-dimers, which may explain why the analytical sensitivity is not higher. Optimally-designed PCR tests typically achieve single-molecule sensitivity with plasmid controls in the absence of interference from background DNA. In addition, the 3’ -CC of CCoV (M)-R is complementary to the 3’ -GG of CDV(N)-F and CPV(VP2)-P, although in the latter case, depending on the exact placing of the BHQ3, dimer-formation may be blocked by the quencher.

Response: The Oligo 7.0 software was used to design the specific primers and probes. After determination of the locations and sequences of primers and probes, the Tm values are obtained automatically using the Oligo 7.0 software. The software has been stated in Lines 134-135 in the revised manuscript.

     The multiple sequence alignments were compared to find conserved regions of the sequences using the MEGA-X software. The conserved regions of the CCoV M gene, CRV VP7 gene, CPV VP2 gene, and CDV N gene were selected as targeted regions to design the four pairs of specific primers and corresponding probes. The software has been stated in Lines 131-132 in the revised manuscript.

The principle of designing primer and probe is to select the conserved region of the viral genome as targeting regions, and design primers and probes that are complementary to the template gene sequences; the lengths of primer and probe are set between 20 and 30 bp; the energy at the 5' end of the primer and probe is higher than that at the 3' end; strive to achieve as much as possible that no hairpin loop formation is formed inside the primer or probe, and no primer dimer is formed between primer and probe, between probe and probe, or between primer and probe. However, due to the involvement of multiple pairs of primers and probes in multiplex RT-qPCR, it is very difficult to achieve the above requirements fully. Therefore, we need to try and optimize various reaction conditions such as primer and probe concentration, annealing temperature, and reaction cycle to obtain the optimal reaction conditions, and establish a specific and sensitive multiplex RT-qPCR. Especially for primer hairpin loop formation and primer/primer, primer/probe and probe/probe dimers, it might be solved by adjusting the annealing temperature.

The above information has been added in the revised manuscript. Please see Lines 122-150 in the revised manuscript.

7. Figure 1. Multiple sequence alignments. The font size is very small, and the figure will very likely become unreadable in the final printed version. Since many of the sequences are identical, I suggest removing sequence duplicates from the alignments. The accession numbers of these may be mentioned in the figure text. It will probably be advantageous to make four separate figures. With the reduced number of lines, it should be possible to put each figure in landscape format, which would allow an acceptable font size. Consider also removing the column with the strain name. The accession number is enough.

It would also be an advantage to add a few (3 – 5) sequences from the most closely related virus types; this would allow the reader to evaluate the possibility of cross-reactions. If there is insufficient space, a second series of figures would be appropriate.

Response: The multiple sequence alignments has been compared again. The number of gene sequences has been decreased to remove sequence duplicates from the alignments. The multiple sequence alignments of 4 different viruses are still be put in one Figure instead of dividing into four Figures, so that only one image title can be used instead of four image titles.

In revised Figure 1, only the accession numbers have been retained, and the virus names have been deleted. The virus names and related information have been transferred to Supplementary Table S1 to S4.

Please see Figure 1 and Supplementary Table S1 to S4 in the revised manuscript.

8. L154-164 and L208-216. Plasmid constructs. L208-216 is a much better methodological description and could with advantage replace much of L154-164 in the methods. This information belongs in the methods, rather than the results.

Response: According to the reviewer’s suggestion, the contents in the part Results (L208-216) have been transferred to the part 2.7. Generation of standard plasmid constructs. Please see Lines 190-198 in the revised manuscript.

9. L205-206. Reference PCR. The reference PCR is central to the study and needs to be described. The description should include (a) the analytical sensitivity of the reaction (if known) (b) the target genes (c) how the assay was implemented. The authors say that it is a multiplex reaction, but imply it is not a quadruplex - so how it was multiplexed in order to detect all four virus types is relevant.

Response: The reference PCR has been described according to the reviewer’s suggestion. Please see Lines 239-249 in the revised manuscript.

10. L250-254. LODs. Three decimal places is unrealistic precision. Express instead as whole numbers or in scientific notation with two significant figures, e.g. 111.440  ≈ 111 ≈ 1.1x10². Elsewhere (e.g the abstract), these figures can be summarised to ca. 10²copies for all four virus types.

Response: According to the reviewer’s suggestion, the LODs have been changed to 1.1×10² in the revised manuscript.

11. 3.7. The range of Ct values observed would be relevant. This would allow the repeatability data to be related to values realistic in clinical samples.

Response: The range of Ct values in clinical samples has been added in the revised manuscript. Please see Lines 324-325 in the revised manuscript.

12. L274-276. Coinfections. This sentence is very difficult to read. It would be better to mention the two most important coinfections and refer to the table for further details.

Response: The sentence has been revised. Please see Lines 321-324 in the revised manuscript.

13. L277-278. Sensitivity and specificity. It is not clearly stated what specificity and sensitivity are calculated relative to. If it is the reference assay, then this sentence belongs in the next paragraph after the positivity rates for the reference assay.

Response: The sentence has been changed to the next paragraph. Please see Lines 331-336 in the revised manuscript.

14. L279-284. How well did the two assays agree in detection of coinfections? This is a more demanding task and thus a better test of assay quality. As the numbers will be small, simple descriptive statistics would be sufficient. It would also be interesting to know if the discrepant samples were concentrated among the coinfections.

Response: The positivity rates of co-infections using two assays have been added in Table 5. Please see Table 5 in the revised manuscript.

15. L283 and elsewhere. ‘reported reference assay’. Just ‘reference assay’ would be better.

Response: Revise “reported reference assay” to “reference assay”. Please see Line 331 in the revised manuscript.

16. L289-297; 298-303. This information is in the tables and does not need to be repeated. Table text should be reserved for notes to explain the text, e.g. abbreviations and symbols.

Response: The notes in Table 6 has been deleted. The notes in Table 7 are still retained but revised. Please see Table 6 and Table 7 in the revised manuscript.

17. Table 6, Table 7 and elsewhere. ‘The established multiplex RT-qPCR’; The reference multiplex RT-qPCR’. These are rather cumbersome terms. Why not ‘(the) current PCR’; (the) reference PCR’?

Response: “The current RT-qPCR” and “The reference RT-qPCR” have been used un Table 6 and Table 7. Please see Table 6 and Table 7 in the revised manuscript.

18. 311-319. This belongs in the introduction, where much of what is written has already been said. The section on previously developed tests should also mention other detection methods (serology, cultivation) if they exist. This would be a good place for a general description of the reference PCR. A point which the authors seem to have overlooked is that the reason no quadruplex PCR has previously been developed is that many real time PCR systems are incapable of 4-channel multiplexing. This also places limitations on the implementation of this test in other laboratories, as these instruments are still not standard equipment, and are quite expensive. The authors should mention this, and perhaps suggest ways of implementing their test on simpler instruments.

Response: The contents repeated to those in the Introduction have been deleted. Please see Lines 364-369 in the revised manuscript.

This limitations on the implementation of qPCR have been analysed briefly in the revised manuscript. Please see Lines 372-379 in the revised manuscript.

The general description of the reference PCR has been described briefly in the revised manuscript. Please see Lines 388-394 in the revised manuscript.

 

19. Repeatability. This would be more easily grasped if expressed as a range of C_tvalues and should be accompanied by the number of target copies. It occurs to me that the authors’ LOD experiments would afford (intra-assay) repeatability for copy numbers near 10².

Response: The range of C_t values and the number of target copies have been added to Table 4. Please see Table 4 in the revised manuscript.

 

Comments on the Quality of English Language

20. The standard of English is generally satisfactory throughout. 

Response: Thanks very much!

 

Reviewer #2

Comments and Suggestions for Authors

This is a well-constructed study, that provides analytical and clinical validation of a multiplex PCR approach for 4 different canine viruses.  

Major comments:

1. It would be of interest to know (and be very informative) whether the assay has different sensitivities/specificities based on the collection approach (feces vs. anal swab vs. nasal swab). 

 Response: The recombinant standard plasmid constructs were used to evaluate the sensitivity and specificity of the developed quadruplex RT-qPCR, and it showed high sensitivity and excellent specificity. We are sorry that we did not evaluate the sensitivity and specificity basing on the collection approach (feces vs. anal swab vs. nasal swab). 

Minor comments:

2. I don't think it is necessary to have 3 decimal points for copy number - see lines, abstract as well as lines 251-254, 260-262, 323-324

 Response: The 3 decimal points for copy number have been deleted, and the LODs have been described as 1.1 × 10² in the revised manuscript.

 Comments on the Quality of English Language

Minor comments:

 

3. Line 14: suggest "confirmed to have"

Response: Revise "confirmed to be" to "confirmed to have". Please see Line 14 in the revised manuscript.

4. Line 16: damage

Response: Revise "damages" to "damage". Please see Line 16 in the revised manuscript.

5. Line 71: What do the authors mean by: "uneasy contamination"?

Response: Revise "uneasy contamination" to "reduced risk of contamination". Please see Line 72 in the revised manuscript.

6. Line 333: tha – the

Response: Revise “tha” to “the”. Please see Line 399 in the revised manuscript.

 

7.  Table 6, “Sensitivity” should be on a single line

Response: Done. Please see Table 6 in the revised manuscript.

8. Please check reference #3.

Response: The reference #3 has been revised. Please see Line 497 in the revised manuscript.

 

Best regards,

 

Kaichuang Shi

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a well-constructed study, that provides analytical and clinical validation of a multiplex PCR approach for 4 different canine viruses.  

Major comments:

1. It would be of interest to know (and be very informative) whether the assay has different sensitivities/specificities based on the collection approach (feces vs. anal swab vs. nasal swab). 

 

Minor comments:

1. I don't think it is necessary to have 3 decimal points for copy number - see lines, abstract as well as lines 251-254, 260-262, 323-324

 

 

Comments on the Quality of English Language

Minor comments:

Minor English suggestions:

 

Line 14: suggest "confirmed to have"

Line 16: damage

Line 71: What do the authors mean by: "uneasy contamination"?

Line 333: tha – the

 

able 6, “Sensitivity” should be on a single line

Please check reference #3.

 

Author Response

Revision Notes

April 27, 2024

Dear editor,

The manuscript has been revised carefully according to the editor’s suggestions and the reviewers’ suggestions. The details are as follows.

 

Reviewer #1

Comments and Suggestions for Authors

1. L24-25. Unrealistic precision. 10²is better.

Response: The LODs have been changed to “1.1 × 10²”. Please see Line 25 in the revised manuscript.

2. L45. Popular -> frequent.

Response: “popular” has been revised to “frequent”. Please see Line 46 in the revised manuscript.

3. L71. ‘uneasy contamination’ - > ‘reduced risk of contamination’ 

Response: “uneasy contamination” has been revised to “reduced risk of contamination”. Please see Line 72 in the revised manuscript.

4. L87. First sentence. ‘This study did not involve any experiments on live animals’ would be a better phrasing.

Response: The first sentence has been changed to “This study did not involve any experiments on live animals”. Please see Lines 89-90 in the revised manuscript.

5. L102-106. Clinical samples. This description is rather brief. What kind of sample were they and in what form were they supplied?

Response: The clinical samples include feces, anal and nasal swabs from each dog with different signs of gastroenteritis. All samples were placed in a specialized medical ice box, and transported to our laboratory under ≤4 ℃ condition within 12 h after collection. This information has been added in the revised manuscript. Please see Lines 111-120 in the manuscript.

6. L116-126. What was the procedure for selection of primers? If any form of primer design/selection software was used, this should be stated. If, as appears to be the case, the authors merely searched the multiple sequence alignments for conserved sequences and then adjusted the length of the primers until they got the required Tm, then they should state this. Also, which method was used for determining Tm?

The authors may be interested to know that calculated Tm values may differ quite a lot from the true value: see Pedersen BN, Jenkins A, Paulsen KM, Basset C, Andreassen ÅK. Development of a real-time PCR method for detection of European and Siberian subtype of tick-borne encephalitis virus. Microbiol Res, 2023, 14, 1545–1560. https://doi.org/10.3390/microbiolres14040106

 The software used for generating multiple sequence alignments should be stated.

Looking at the primer sequences, I notice some design errors. CCoV(M)-F has the self-complementary sequence -AATT at the 3’ end, and CRV (VP7)-F has -GC. Both of these are likely to form primer-dimers, which may explain why the analytical sensitivity is not higher. Optimally-designed PCR tests typically achieve single-molecule sensitivity with plasmid controls in the absence of interference from background DNA. In addition, the 3’ -CC of CCoV (M)-R is complementary to the 3’ -GG of CDV(N)-F and CPV(VP2)-P, although in the latter case, depending on the exact placing of the BHQ3, dimer-formation may be blocked by the quencher.

Response: The Oligo 7.0 software was used to design the specific primers and probes. After determination of the locations and sequences of primers and probes, the Tm values are obtained automatically using the Oligo 7.0 software. The software has been stated in Lines 134-135 in the revised manuscript.

     The multiple sequence alignments were compared to find conserved regions of the sequences using the MEGA-X software. The conserved regions of the CCoV M gene, CRV VP7 gene, CPV VP2 gene, and CDV N gene were selected as targeted regions to design the four pairs of specific primers and corresponding probes. The software has been stated in Lines 131-132 in the revised manuscript.

The principle of designing primer and probe is to select the conserved region of the viral genome as targeting regions, and design primers and probes that are complementary to the template gene sequences; the lengths of primer and probe are set between 20 and 30 bp; the energy at the 5' end of the primer and probe is higher than that at the 3' end; strive to achieve as much as possible that no hairpin loop formation is formed inside the primer or probe, and no primer dimer is formed between primer and probe, between probe and probe, or between primer and probe. However, due to the involvement of multiple pairs of primers and probes in multiplex RT-qPCR, it is very difficult to achieve the above requirements fully. Therefore, we need to try and optimize various reaction conditions such as primer and probe concentration, annealing temperature, and reaction cycle to obtain the optimal reaction conditions, and establish a specific and sensitive multiplex RT-qPCR. Especially for primer hairpin loop formation and primer/primer, primer/probe and probe/probe dimers, it might be solved by adjusting the annealing temperature.

The above information has been added in the revised manuscript. Please see Lines 122-150 in the revised manuscript.

7. Figure 1. Multiple sequence alignments. The font size is very small, and the figure will very likely become unreadable in the final printed version. Since many of the sequences are identical, I suggest removing sequence duplicates from the alignments. The accession numbers of these may be mentioned in the figure text. It will probably be advantageous to make four separate figures. With the reduced number of lines, it should be possible to put each figure in landscape format, which would allow an acceptable font size. Consider also removing the column with the strain name. The accession number is enough.

It would also be an advantage to add a few (3 – 5) sequences from the most closely related virus types; this would allow the reader to evaluate the possibility of cross-reactions. If there is insufficient space, a second series of figures would be appropriate.

Response: The multiple sequence alignments has been compared again. The number of gene sequences has been decreased to remove sequence duplicates from the alignments. The multiple sequence alignments of 4 different viruses are still be put in one Figure instead of dividing into four Figures, so that only one image title can be used instead of four image titles.

In revised Figure 1, only the accession numbers have been retained, and the virus names have been deleted. The virus names and related information have been transferred to Supplementary Table S1 to S4.

Please see Figure 1 and Supplementary Table S1 to S4 in the revised manuscript.

8. L154-164 and L208-216. Plasmid constructs. L208-216 is a much better methodological description and could with advantage replace much of L154-164 in the methods. This information belongs in the methods, rather than the results.

Response: According to the reviewer’s suggestion, the contents in the part Results (L208-216) have been transferred to the part 2.7. Generation of standard plasmid constructs. Please see Lines 190-198 in the revised manuscript.

9. L205-206. Reference PCR. The reference PCR is central to the study and needs to be described. The description should include (a) the analytical sensitivity of the reaction (if known) (b) the target genes (c) how the assay was implemented. The authors say that it is a multiplex reaction, but imply it is not a quadruplex - so how it was multiplexed in order to detect all four virus types is relevant.

Response: The reference PCR has been described according to the reviewer’s suggestion. Please see Lines 239-249 in the revised manuscript.

10. L250-254. LODs. Three decimal places is unrealistic precision. Express instead as whole numbers or in scientific notation with two significant figures, e.g. 111.440  ≈ 111 ≈ 1.1x10². Elsewhere (e.g the abstract), these figures can be summarised to ca. 10²copies for all four virus types.

Response: According to the reviewer’s suggestion, the LODs have been changed to 1.1×10² in the revised manuscript.

11. 3.7. The range of Ct values observed would be relevant. This would allow the repeatability data to be related to values realistic in clinical samples.

Response: The range of Ct values in clinical samples has been added in the revised manuscript. Please see Lines 324-325 in the revised manuscript.

12. L274-276. Coinfections. This sentence is very difficult to read. It would be better to mention the two most important coinfections and refer to the table for further details.

Response: The sentence has been revised. Please see Lines 321-324 in the revised manuscript.

13. L277-278. Sensitivity and specificity. It is not clearly stated what specificity and sensitivity are calculated relative to. If it is the reference assay, then this sentence belongs in the next paragraph after the positivity rates for the reference assay.

Response: The sentence has been changed to the next paragraph. Please see Lines 331-336 in the revised manuscript.

14. L279-284. How well did the two assays agree in detection of coinfections? This is a more demanding task and thus a better test of assay quality. As the numbers will be small, simple descriptive statistics would be sufficient. It would also be interesting to know if the discrepant samples were concentrated among the coinfections.

Response: The positivity rates of co-infections using two assays have been added in Table 5. Please see Table 5 in the revised manuscript.

15. L283 and elsewhere. ‘reported reference assay’. Just ‘reference assay’ would be better.

Response: Revise “reported reference assay” to “reference assay”. Please see Line 331 in the revised manuscript.

16. L289-297; 298-303. This information is in the tables and does not need to be repeated. Table text should be reserved for notes to explain the text, e.g. abbreviations and symbols.

Response: The notes in Table 6 has been deleted. The notes in Table 7 are still retained but revised. Please see Table 6 and Table 7 in the revised manuscript.

17. Table 6, Table 7 and elsewhere. ‘The established multiplex RT-qPCR’; The reference multiplex RT-qPCR’. These are rather cumbersome terms. Why not ‘(the) current PCR’; (the) reference PCR’?

Response: “The current RT-qPCR” and “The reference RT-qPCR” have been used un Table 6 and Table 7. Please see Table 6 and Table 7 in the revised manuscript.

18. 311-319. This belongs in the introduction, where much of what is written has already been said. The section on previously developed tests should also mention other detection methods (serology, cultivation) if they exist. This would be a good place for a general description of the reference PCR. A point which the authors seem to have overlooked is that the reason no quadruplex PCR has previously been developed is that many real time PCR systems are incapable of 4-channel multiplexing. This also places limitations on the implementation of this test in other laboratories, as these instruments are still not standard equipment, and are quite expensive. The authors should mention this, and perhaps suggest ways of implementing their test on simpler instruments.

Response: The contents repeated to those in the Introduction have been deleted. Please see Lines 364-369 in the revised manuscript.

This limitations on the implementation of qPCR have been analysed briefly in the revised manuscript. Please see Lines 372-379 in the revised manuscript.

The general description of the reference PCR has been described briefly in the revised manuscript. Please see Lines 388-394 in the revised manuscript.

 

19. Repeatability. This would be more easily grasped if expressed as a range of C_tvalues and should be accompanied by the number of target copies. It occurs to me that the authors’ LOD experiments would afford (intra-assay) repeatability for copy numbers near 10².

Response: The range of C_t values and the number of target copies have been added to Table 4. Please see Table 4 in the revised manuscript.

 

Comments on the Quality of English Language

20. The standard of English is generally satisfactory throughout. 

Response: Thanks very much!

 

Reviewer #2

Comments and Suggestions for Authors

This is a well-constructed study, that provides analytical and clinical validation of a multiplex PCR approach for 4 different canine viruses.  

Major comments:

1. It would be of interest to know (and be very informative) whether the assay has different sensitivities/specificities based on the collection approach (feces vs. anal swab vs. nasal swab). 

 Response: The recombinant standard plasmid constructs were used to evaluate the sensitivity and specificity of the developed quadruplex RT-qPCR, and it showed high sensitivity and excellent specificity. We are sorry that we did not evaluate the sensitivity and specificity basing on the collection approach (feces vs. anal swab vs. nasal swab). 

Minor comments:

2. I don't think it is necessary to have 3 decimal points for copy number - see lines, abstract as well as lines 251-254, 260-262, 323-324

 Response: The 3 decimal points for copy number have been deleted, and the LODs have been described as 1.1 × 10² in the revised manuscript.

 Comments on the Quality of English Language

Minor comments:

 

3. Line 14: suggest "confirmed to have"

Response: Revise "confirmed to be" to "confirmed to have". Please see Line 14 in the revised manuscript.

4. Line 16: damage

Response: Revise "damages" to "damage". Please see Line 16 in the revised manuscript.

5. Line 71: What do the authors mean by: "uneasy contamination"?

Response: Revise "uneasy contamination" to "reduced risk of contamination". Please see Line 72 in the revised manuscript.

6. Line 333: tha – the

Response: Revise “tha” to “the”. Please see Line 399 in the revised manuscript.

 

7.  Table 6, “Sensitivity” should be on a single line

Response: Done. Please see Table 6 in the revised manuscript.

8. Please check reference #3.

Response: The reference #3 has been revised. Please see Line 497 in the revised manuscript.

 

Best regards,

 

Kaichuang Shi

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed the issues I raised in my previous review, but on re-reading the manuscript, I have noticed some additional issues that need to be addressed, as well as some textual matters which could be improved. 

Abstract.

L11 - L12. Delete from beginning, up to 'canine coronavirus...'. 

L13. Delete 'infected' 

L24 'with other canine viruses'  -> 'with the n other canine viruses tested' .  

Testing analytical specificity is always challenging as clinical samples may contain an enormous diversity of microorganisms. The possibility of cross-reaction to some harmless microbe that nobody knows about is alway present.  Thus, it is almost impossible to prove absence of cross-reaction and it remains part of quality assurance for the rest of the life of the test. The rationale for choosing the present cross-reaction panel should be outlined in the discussion and any weaknesses should be addressed.  

L25. 25 Delete 'for CCoV ... respectively. Consider substituting 'for all four types'. 

Introduction. 

L39. Delete 'vomiting and diarrhea. That is what gastroenteritis is. 

L45. 'include' -> 'are' 

L75/76. 'is necessary' -> would be useful 

L79. 'In this study we have developed and evaluated a quadruplex...' would be better. 

Materials and methods 

L89. Delete 'different' 

L110-111. 'A total of 1,028 clinical samples, including feces and anal and nasal swabs from each dog, were obtained from 1,028 sick dogs' 

This is incorrect. Feces, anal swab and nasal swab from each of 1028 dogs = 1028 x 3 = 3084 samples. How many dogs and how many samples of each type are important methodological parameters. They must be correctly and explicitly stated. 

L114. 'under ≤4 ℃ condition within 12 h after collection' -> 'at ≤4 ℃ within 12 h of collection' 

L120. 'The 36' -> 'Thirty-six'. Although arabic numerals should not be used at the beginning of a sentence, the word for the number is entirely permissible. 

L128. Delete 'of the sequences' 

L129-130. 'The conserved regions ... N-gene' -> 'These' 

L133-139. Consider moving this to the introduction as criteria for primer-design. 

L141-2. 'These primers...' This sentence is unnecessary. 

L145-147. How was this done? Performing a BLAST search for cross-reaction is a tricky business as a certain degree of mismatch is tolerated in PCR and whether or not cross-reaction occurs depends on the number of mismatches, their type and their distance from the 3' end. The parameter settings in BLAST also have a big effect, particularly for short sequences such as primers. Thus, this part should be carefully described and the results reported, or it should be omitted altogether.  

L172. Delete 'clinical'. Also, please specify the (approximate) amount of feces used.   

L206. '56' -> '51-61' 

Section 2.13. Reverse the order of the two paragraphs and re-word as appropriate. This will bring the evaluation of clinical sensitivity and specificity to its natural position, after the two assays to be compared have been described. 

Results 

Section 3.1. Delete the first sentence and move the rest to the appropriate part of the materials and methods.

L253-5. This sentence belongs in the materials and methods. Also, please outline how orthogonal experimental design/analysis was implemented and/or provide a reference. This is not common knowledge in the PCR world. 

Figure 2. Please include a definition of delta-Rn in the figure text. 'Background-corrected fluorescence' is my suggestion. 

L285-290. Remove (2) spelling errors. The text could be usefully shortened by beginning the sentence with 'The LODs and their 95% confidence intervals...', which would allow each LOD to be expressed as (e.g) '109 (98-127)'. An alternative abbreviation is 'CI₉₅ = '. The latter could be usefully used in  

L307-8. Delete the first sentence and begin the 2nd sentence with 'the positivity rates for clinical samples with the established assay were. Use the same formulation for 315-316. 

L329 - 337. This merely repeats the information in Table 6. Delete. 

Discussion 

L348. Add 'as well as appropriate instrumentation' to the end of the sentence. 

L349. Insert 'suitable' before qPCR. 

L354. Delete 'strong' and 'only'. These adjectives are not justified by the limited cross-reactivity testing performed. 

358-365. Delete, and alter the following sentence accordingly. 

This information is adequately presented elsewhere in the text and does not need to be repeated here. 

372-end. Reduce this to a brief discussion of whether the infection levels found are similar to those found elsewhere. 2-3 sentences should be quite sufficient. 

This is a methodological paper and, as such, not an appropriate place for a lengthy discussion of the epidemiology and pathology of canine gastroenteritis or its zoonotic potential. 

 

 

Comments on the Quality of English Language

The English is of a general high standard and does not need to be improved except in the specific cases I have indicated. 

Author Response

Revision Notes

May 2, 2024

 

Dear editor,

We have revised our manuscript carefully according to the reviewer's suggestions. The details are as follows.

 

Comments and Suggestions for Authors

The authors have addressed the issues I raised in my previous review, but on re-reading the manuscript, I have noticed some additional issues that need to be addressed, as well as some textual matters which could be improved.

 

Abstract.

 

1. L11 - L12. Delete from beginning, up to 'canine coronavirus...'.

Response: The content has been deleted. Please see Lines 11-12 in the revised manuscript.

 

2. L13. Delete 'infected'

Response: Done. Please see Line 13 in the revised manuscript.

 

3. L24 'with other canine viruses' -> 'with the n other canine viruses tested' .

Response: Revise “with other canine viruses” to “with the other canine viruses tested”. Please see Line 24 in the revised manuscript.

 

4. Testing analytical specificity is always challenging as clinical samples may contain an enormous diversity of microorganisms. The possibility of cross-reaction to some harmless microbe that nobody knows about is always present. Thus, it is almost impossible to prove absence of cross-reaction and it remains part of quality assurance for the rest of the life of the test. The rationale for choosing the present cross-reaction panel should be outlined in the discussion and any weaknesses should be addressed.

Response: This issue was discussed in the part Discussion. Please see Lines 386-392 in the revised manuscript.

 

5. L25. 25 Delete 'for CCoV ... respectively. Consider substituting 'for all four types'.

Response: Revise “CCoV, CRV, CPV, and CDV, respectively” to “all four plasmid constructs”. Please see Line 25 in the revised manuscript.

 

Introduction.

 

6. L39. Delete 'vomiting and diarrhea. That is what gastroenteritis is.

Response: Delete “vomiting and diarrhea”. Please see Lines 40-41 in the revised manuscript.

 

7. L45. 'include' -> 'are'

Response: Revise “include” to “are”. Please see Line 46 in the revised manuscript.

 

8. L75/76. 'is necessary' -> would be useful

Response: Revise “is necessary” to “would be useful”. Please see Line 76-77 in the revised manuscript.

 

9. L79. 'In this study we have developed and evaluated a quadruplex...' would be better.

Response: This sentence has been revised to “In this study, we have developed and evaluated a quadruplex...”. Please see Line 80 in the revised manuscript.

 

Materials and methods

 

10. L89. Delete 'different'

Response: Delete “different”. Please see Line 90 in the revised manuscript.

 

11. L110-111. 'A total of 1,028 clinical samples, including feces and anal and nasal swabs from each dog, were obtained from 1,028 sick dogs'

This is incorrect. Feces, anal swab and nasal swab from each of 1028 dogs = 1028 x 3 = 3084 samples. How many dogs and how many samples of each type are important methodological parameters. They must be correctly and explicitly stated.

Response: In our study, the feces, and anal and nasal swabs from each dog were mixed together in one tube, votexed, then 200 μL supernatant was used to test viruses. Therefore, the mixture of the feces, and anal and nasal swabs from each dog was considered as one sample.

The sentence has been revised as follows (Please see Lines 111-114 in the revised manuscript):

A total of 1,028 clinical samples (the samples of feces, and anal and nasal swabs from each dog were mixed together in one tube for test of viruses, so the mixture of the feces, and anal and nasal swabs from the same dog was considered as a sample) were obtained from 1,028 sick dogs with different signs of gastroenteritis.

 

12. L114. 'under ≤4 ℃ condition within 12 h after collection' -> 'at ≤4 ℃ within 12 h of collection'

Response: Revise “under ≤4 ℃ condition within 12 h after collection” to “at ≤4 ℃ within 12 h of collection”. Please see Line 117 in the revised manuscript.

 

13. L120. 'The 36' -> 'Thirty-six'. Although arabic numerals should not be used at the beginning of a sentence, the word for the number is entirely permissible.

Response: Revise “The 36” to “'Thirty-six”. Please see Line 123 in the revised manuscript.

 

14. L128. Delete 'of the sequences'

Response: Delete “of the sequences”. Please see Line 131 in the revised manuscript.

 

15. L129-130. 'The conserved regions ... N-gene' -> 'These'

Response: Revise “The conserved regions ... N-gene” to “These”. Please see Lines 132-133 in the revised manuscript.

 

16. L133-139. Consider moving this to the introduction as criteria for primer-design.

Response: This paragraph is described for designing the primers and probes. Considering the content and coherence of the previous and subsequent sections, it is appropriate to place the principles of designing primer and probe design in this paragraph. Therefore, this content is still retained here. Please see Lines 136-142 in the revised manuscript.

 

17. L141-2. 'These primers...' This sentence is unnecessary.

Response: Delete this sentence. Please see Lines 144-145 in the revised manuscript.

 

18. L145-147. How was this done? Performing a BLAST search for cross-reaction is a tricky business as a certain degree of mismatch is tolerated in PCR and whether or not cross-reaction occurs depends on the number of mismatches, their type and their distance from the 3' end. The parameter settings in BLAST also have a big effect, particularly for short sequences such as primers. Thus, this part should be carefully described and the results reported, or it should be omitted altogether.

Response: Really, performing a BLAST search for cross-reaction is a tricky business. Therefore, this sentence has been deleted according to the reviewer’s suggestion. Please see Lines 148-150 in the revised manuscript.

 

19. L172. Delete 'clinical'. Also, please specify the (approximate) amount of feces used.

Response: Delete “clinical”. In addition, the amount of feces used in this study has been explained in the issue 11 above. Please see Lines 111-114 in the revised manuscript.

 

20. L206. '56' -> '51-61'

Response: Revise “56” to “51-61”. Please see Line 212 in the revised manuscript.

 

21. Section 2.13. Reverse the order of the two paragraphs and re-word as appropriate. This will bring the evaluation of clinical sensitivity and specificity to its natural position, after the two assays to be compared have been described.

Response: The order of the two paragraphs has been reversed according to the reviewer’s suggestion. Please see Lines 234-253 in the revised manuscript.

 

Results

 

22. Section 3.1. Delete the first sentence and move the rest to the appropriate part of the materials and methods.

Response: Delete the first sentence, and the rest of the paragraph has been moved to the part of “2.7. Generation of standard plasmid constructs” in the Materials and Methods. Please Lines 198-200 and Lines 255-260 in the revised manuscript.

 

23. L253-5. This sentence belongs in the materials and methods. Also, please outline how orthogonal experimental design/analysis was implemented and/or provide a reference. This is not common knowledge in the PCR world.

Response: This sentence has been revised. Please see Line 262-264 in the revised manuscript.

In addition, experiments for acquiring the optimal conditions were performed on the ABI QuantStudio 5 qPCR system (ABI, Carlsbad, CA, USA) by adjusting the annealing temperature (55 °C-61 °C), primer and probe concentrations (200 pmol/µL, 0.2-0.6 µL), and reaction cycles (25-45 cycles). This has been described in the part “2.8. Determination of the reaction conditions”. Please see Lines 203-205 in the revised manuscript.

Since the first sentence in “3.21. Determination of the reaction conditions” has been revised, and the experiments for acquiring the optimal conditions have been described in “2.8. Determination of the reaction conditions”, the words “orthogonal experiments” has been deleted. Please see Line 262-264 in the revised manuscript.

 

24. Figure 2. Please include a definition of delta-Rn in the figure text. 'Background-corrected fluorescence' is my suggestion.

Response: These contents have been described in the Figure 2 caption. Please see Lines 278-282 in the revised manuscript.

 

25. L285-290. Remove (2) spelling errors. The text could be usefully shortened by beginning the sentence with 'The LODs and their 95% confidence intervals...', which would allow each LOD to be expressed as (e.g) '109 (98-127)'. An alternative abbreviation is 'CI95 = '. The latter could be usefully used in

Response: The sentence has been revised according to the reviewer’s suggestion. Please see Lines 297-302.

 

26. L307-8. Delete the first sentence and begin the 2nd sentence with 'the positivity rates for clinical samples with the established assay were. Use the same formulation for 315-316.

Response: Delete the first sentence of the two paragraphs, respectively, according to the reviewer’s suggestions. Please see Lines 319-331 in the revised manuscript.

 

27. L329 - 337. This merely repeats the information in Table 6. Delete.

Response: Delete these contents. Please see Lines 343-351 in the revised manuscript.

 

Discussion

 

28. L348. Add 'as well as appropriate instrumentation' to the end of the sentence.

Response: Done. Please see Lines 362-363 in the revised manuscript.

 

29. L349. Insert 'suitable' before qPCR.

Response: Done. Please see Line 360 in the revised manuscript.

 

30. L354. Delete 'strong' and 'only'. These adjectives are not justified by the limited cross-reactivity testing performed.

Response: Delete “strong” and “only”. Please see Line 369 in the revised manuscript.

 

31. 358-365. Delete, and alter the following sentence accordingly.

This information is adequately presented elsewhere in the text and does not need to be repeated here.

Response: Delete these sentences have been deleted according to the reviewer’s suggestion. Please see Lines 375-381 in the revised manuscript.

 

32. 372-end. Reduce this to a brief discussion of whether the infection levels found are similar to those found elsewhere. 2-3 sentences should be quite sufficient.

This is a methodological paper and, as such, not an appropriate place for a lengthy discussion of the epidemiology and pathology of canine gastroenteritis or its zoonotic potential.

Response: According to the reviewer’s suggestion, the last two paragraphs have been re-written. Please see Lines 393-467 in the revised manuscript.

 

Comments on the Quality of English Language

33. The English is of a general high standard and does not need to be improved except in the specific cases I have indicated.

Response: We have revised the manuscript carefully according to the reviewer’s suggestions. Please see the revised manuscript.

 

Best regards,

 

Kaichuang Shi

Author Response File: Author Response.pdf