Detection of Cassava Mosaic Disease and Assessment of Selected Agronomic Traits of Cassava (Manihot esculenta)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

"The Nuru phenotype tool was inefficient in detecting CMD in plants derived from cuttings obtained from asymptomatic mother plants at the latent phase of development, but efficiently detected CMD similar to the molecular analytic technique for plants derived from cuttings obtained from symptomatic mother plants. These findings partly agree with Johansen et al. [50], Arakpogun et al. [51], and Cuellar et al. [25], who opined that phenotype tools such as the Nuru app are efficient for phenotypic detection of plant diseases like molecular analysis techniques".

Although the authors tried to juxtapose their findings with those of previous studies using the Nuru phenotype tool as seen in the above paragraph.

I would suggest that the authors provide a brief paragraph on the working principles of the Nuru app. This would probably justify why the app is inefficient for phenotypical detection of CMD 41 at 3 MAP.   

This could be in the materials and methods section or the discussion. 

 

Author Response

Njala University (NU),

Njala Campus, Sierra Leone,

26^th April, 2025.

 

Dear Editor,

It is my pleasure to resubmit the revised version of the manuscript plants-3502534 entitled “Identification and Distribution of Begomoviruses Infecting Cassava Fields in Sierra Leone”. The manuscript has been revised as suggested by the Reviewer and corrections can be traced in red font where appropriate. The responses to the reviewer’s comments are below. We hope to hear from you about this manuscript soon.

 

Best regards,

Musa Decius Saffa

m.saffa21@yahoo.com

REVIEWER 1

Comments and Suggestions for Authors

"The Nuru phenotype tool was inefficient in detecting CMD in plants derived from cuttings obtained from asymptomatic mother plants at the latent phase of development, but efficiently detected CMD similar to the molecular analytic technique for plants derived from cuttings obtained from symptomatic mother plants. These findings partly agree with Johansen et al. [50], Arakpogun et al. [51], and Cuellar et al. [25], who opined that phenotype tools such as the Nuru app are efficient for phenotypic detection of plant diseases like molecular analysis techniques".

Although the authors tried to juxtapose their findings with those of previous studies using the Nuru phenotype tool as seen in the above paragraph.

Reviewer #1 Comments: I would suggest that the authors provide a brief paragraph on the working principles of the Nuru app. This would probably justify why the app is inefficient for phenotypical detection of CMD 41 at 3 MAP.   

This could be in the materials and methods section or the discussion. 

Response: Thanks for the inputs. The corrections have been done as suggested. The correction can be traced on lines 118-126.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper by Musa Decius Saffa et al., titled "Detection of Cassava Mosaic Disease and Assessment of Selected Agronomic Traits of Cassava (Manihot esculenta)", presents a methodologically rigorous investigation into CMD detection efficacy and its impact on cassava agronomic traits across Sierra Leone's major cassava-producing regions. The study effectively combines phenotypic screening via the Nuru App and molecular virus indexing to assess disease prevalence and varietal resistance. Notably, it identifies the SLICASS 4 variety as CMD-resistant, corroborated by both phenotypic absence of symptoms and negative molecular results. The integration of multilocational field trials strengthens the findings, revealing that CMD incidence and whitefly pressure vary significantly by region and cutting origin. Statistical robustness is evident in the use of ANOVA and DMRT.

However, while the study excels in empirical depth, the introduction could be streamlined. In line 63, it is recommended that the introduction begins by stating that CMD is a disease caused by 11 virus species, then narrows down to the specific strains relevant to the study region. Additionally, including a brief explanation of CMD symptoms—such as leaf chlorosis, distortion, and stunting—is essential, especially since one of the study’s aims involves evaluating a phenotypic detection tool.

In the materials and methods section, it would be beneficial to mention the source of the stem cuttings used in the experiments, as this information is critical for reproducibility.

The discussion effectively contextualizes results within CMD epidemiology and had clear information and references. However, caution should be applied in line 308, where resistance is incorrectly attributed to phenotypic studies. Resistance is not a phenotypic trait; rather, it is confirmed through molecular techniques as indication of virus replication which was clearly described in the discussion for that the sentence should be revised for clarity.

Similarly, line 318 should be rephrased by removing the last three words. The scientifically accurate portion is: "Disease diagnosis limited to the visual or phenotypic assessment of symptoms on plants is insufficient for determination of its resistance."

Regarding the conclusion, there is a discrepancy between the discussion and the final statements. In the discussion, the authors partially agree with the Nuru app's utility for detecting plant diseases at early infection stages (line 367), but the conclusion reports a contrasting statement. This should be corrected to align with the discussion, emphasizing that the Nuru app has limitations in detecting latent infections but performs comparably to molecular tools at later stages.

The English language is mostly clear but could benefit from editorial polishing for conciseness and flow. Care is needed with abbreviations, as each abbreviation should be defined the first time it appears. A thorough review of the entire text is recommended to ensure consistent abbreviation usage.

 References are current and relevant

The research significantly contributes to CMD management strategies, emphasizing the utility of disease-free planting materials, environmental influence on disease severity, and the potential limitations of AI tools like Nuru in early CMD detection.

In conclusion, this is a significant study that contributes to understanding the spread of CMD in Sierra Leone. Minor revisions are suggested to improve clarity. With these refinements, the paper can have an even greater impact and readability and is recommended for publication

Author Response

Njala University (NU),

Njala Campus, Sierra Leone,

26^th April, 2025.

 

Dear Editor,

It is my pleasure to resubmit the revised version of the manuscript plants-3502534 entitled “Identification and Distribution of Begomoviruses Infecting Cassava Fields in Sierra Leone”. The manuscript has been revised as suggested by the Reviewer and corrections can be traced in red font where appropriate. The responses to the reviewer’s comments are below. We hope to hear from you about this manuscript soon.

 

Best regards,

Musa Decius Saffa

REVIEWER 2

Comments and Suggestions for Authors

The paper by Musa Decius Saffa et al., titled "Detection of Cassava Mosaic Disease and Assessment of Selected Agronomic Traits of Cassava (Manihot esculenta)", presents a methodologically rigorous investigation into CMD detection efficacy and its impact on cassava agronomic traits across Sierra Leone's major cassava-producing regions. The study effectively combines phenotypic screening via the Nuru App and molecular virus indexing to assess disease prevalence and varietal resistance. Notably, it identifies the SLICASS 4 variety as CMD-resistant, corroborated by both phenotypic absence of symptoms and negative molecular results. The integration of multilocational field trials strengthens the findings, revealing that CMD incidence and whitefly pressure vary significantly by region and cutting origin. Statistical robustness is evident in the use of ANOVA and DMRT.

Reviewer #2 Comments: However, while the study excels in empirical depth, the introduction could be streamlined. In line 63, it is recommended that the introduction begins by stating that CMD is a disease caused by 11 virus species, then narrows down to the specific strains relevant to the study region. Additionally, including a brief explanation of CMD symptoms—such as leaf chlorosis, distortion, and stunting—is essential, especially since one of the study’s aims involves evaluating a phenotypic detection tool.

Response: The revision has been done as suggested by the reviewer. It can be traced on lines 66-75.

Reviewer #2 Comments: In the materials and methods section, it would be beneficial to mention the source of the stem cuttings used in the experiments, as this information is critical for reproducibility.

Response: Thanks for this input. The correction has been done as suggested (lines 115-118; 126-132)

Reviewer #2 Comments: The discussion effectively contextualizes results within CMD epidemiology and had clear information and references. However, caution should be applied in line 308, where resistance is incorrectly attributed to phenotypic studies. Resistance is not a phenotypic trait; rather, it is confirmed through molecular techniques as indication of virus replication which was clearly described in the discussion for that the sentence should be revised for clarity.

Response: Thanks for your correction. You are right. The sentence has been corrected accordingly. Resistance has been deleted in the reference sentence (lines 338-339).

Reviewer #2 Comments: Similarly, line 318 should be rephrased by removing the last three words. The scientifically accurate portion is: "Disease diagnosis limited to the visual or phenotypic assessment of symptoms on plants is insufficient for determination of its resistance."

Response: The last three words have been removed as suggested (lines 328-330; 346-348).

Reviewer #2 Comments: Regarding the conclusion, there is a discrepancy between the discussion and the final statements. In the discussion, the authors partially agree with the Nuru app's utility for detecting plant diseases at early infection stages (line 367), but the conclusion reports a contrasting statement. This should be corrected to align with the discussion, emphasizing that the Nuru app has limitations in detecting latent infections but performs comparably to molecular tools at later stages.

Response: The correction has been done as suggested (lines 401-415).

Reviewer #2 Comments: The English language is mostly clear but could benefit from editorial polishing for conciseness and flow. Care is needed with abbreviations, as each abbreviation should be defined the first time it appears. A thorough review of the entire text is recommended to ensure consistent abbreviation usage.

Response: A thorough review of the entire text has been done as suggested.

Reviewer #2 Comments: References are current and relevant

Response: Thanks for this feedback.

Reviewer #2 Comments: The research significantly contributes to CMD management strategies, emphasizing the utility of disease-free planting materials, environmental influence on disease severity, and the potential limitations of AI tools like Nuru in early CMD detection.

Response: Thanks for this feedback.

Reviewer #2 Comments: In conclusion, this is a significant study that contributes to understanding the spread of CMD in Sierra Leone. Minor revisions are suggested to improve clarity. With these refinements, the paper can have an even greater impact and readability and is recommended for publication

Response: Thanks for this feedback. The minor revision has been done as suggested.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Authors Musa Decius Saffa and co-workers presented here a manuscript entitled „Detection of Cassava Mosaic Disease and Assessment of Selected Agronomic Traits of Cassava (Manihot esculenta)“.

The authors used the "PlantVillage Nuru App" to detect viruses causing cassava mosaic disease in Sierra Leone, along with methods for virus indexing, evaluating the number of whiteflies on the crop, and assessing yield parameters of cassava with varying degrees of virus disease infection.

A survey of cassava mosaic disease (CMD) incidence was conducted on 80 farms in 4 regions of Sierra Leone. Field experiments were conducted in the same 4 regions. Cuttings were planted from symptomatic and symptomless plants of the susceptible variety and from the resistant variety. Highly susceptible infected plants were placed at the edges of the field as a source of virus.

 

CMD incidence, number of whiteflies on plants, plant growth and root yield were assessed.

In addition to phenotypic evaluation of symptoms, PCR was used to determine the presence of the virus.

The authors also evaluated the correlations between the different factors observed in the experiment.

The authors found the highest level of CMD symptoms in the variant of the experiment established from symptomatic plants. They did not detect any CMD symptoms in the resistant genotype, nor did they prove the presence of the virus by PCR. The resistant genotype also had the best growth parameters and the highest cassava yield.

The experiments were well established and performed. The data obtained allow to achieve the stated objective of the work, which is to diagnose CMD using the "PlantVillage Nuru App" application and the laboratory diagnostic method of PCR and to compare the agronomic parameters of plants with different degrees of CMD expression.

The authors need to better evaluate the data obtained and draw proper conclusions from it, which according to the manuscript submitted, they have failed to do.

 

In the discussion on lines 305-322, they correctly define the concepts of resistance and tolerance of plants to the pathogen and correctly state that the degree of resistance cannot be determined by symptoms alone, but that a diagnostic method must be used to detect symptomless infection.

It is not appropriate to write on lines 323-324 that the SLICASS 4 genotype is potentially resistant when the results of the paper show that it is indeed resistant to the viruses causing CMD.

Then, on lines 333-339, it is written that plants based on symptomatic and symptomless plants of the susceptible variety are symptomless, i.e. tolerant to infection, when it is clear from the data presented that they had symptoms. It goes without saying that the intensity of symptoms differs in plants already infected at the time of planting from those that were infected from surrounding plants only during the course of the experiment. It is therefore by no means a question of tolerance.

Why do the authors write on line 364 that the application of Nuru was not effective in determining symptoms in symptomless plants when they already write on line 373 that the application was effective?

I miss the results of the CMD prevalence survey described in Section 2.1.

 

I recommend reworking the discussion and interpreting the data correctly.

 

Further comments:

line 39: cocoa – if you mean the name of the variety, it should be capitalized. The same problem appeared on lines 182, 184, 186, 190, 191, 194, 195, 199, 200, 206, 215, 234, 247, 248, 260, 263, 297, 333, 339, 377

line 129: Port Loko District in the Northern province - according to other parts of the text, it is the Northwestern province

line 131: improved released genotype - indicate what the improvement in the genotype is - probably resistance to CMD; and continue to state it further in the text

line 144: The plant height (cm), stem girth (mm), and number of nodes. - the sentence looks incomplete

line 155: For each treatment in the trial - the word treatment is used incorrectly, it should be a variant of experiment; and continue to state it further in the text

line 168: and an additional 10 min cycle at 72 °C. - the added 10 minutes cannot be called a cycle

line 171: 50 ng ultrapure water - this number is probably wrong

Comments on the Quality of English Language

Revising the English will help make the manuscript more readable.

Author Response

Njala University (NU),

Njala Campus, Sierra Leone,

26^th April, 2025.

 

Dear Editor,

It is my pleasure to resubmit the revised version of the manuscript plants-3502534 entitled “Identification and Distribution of Begomoviruses Infecting Cassava Fields in Sierra Leone”. The manuscript has been revised as suggested by the Reviewer and corrections can be traced in red font where appropriate. The responses to the reviewer’s comments are below. We hope to hear from you about this manuscript soon.

 

Best regards,

Musa Decius Saffa

REVIEWER 3

Comments and Suggestions for Authors

Authors Musa Decius Saffa and co-workers presented here a manuscript entitled „Detection of Cassava Mosaic Disease and Assessment of Selected Agronomic Traits of Cassava (Manihot esculenta)“.

The authors used the "PlantVillage Nuru App" to detect viruses causing cassava mosaic disease in Sierra Leone, along with methods for virus indexing, evaluating the number of whiteflies on the crop, and assessing yield parameters of cassava with varying degrees of virus disease infection.

A survey of cassava mosaic disease (CMD) incidence was conducted on 80 farms in 4 regions of Sierra Leone. Field experiments were conducted in the same 4 regions. Cuttings were planted from symptomatic and symptomless plants of the susceptible variety and from the resistant variety. Highly susceptible infected plants were placed at the edges of the field as a source of virus.

CMD incidence, number of whiteflies on plants, plant growth and root yield were assessed.

In addition to phenotypic evaluation of symptoms, PCR was used to determine the presence of the virus.

The authors also evaluated the correlations between the different factors observed in the experiment.

The authors found the highest level of CMD symptoms in the variant of the experiment established from symptomatic plants. They did not detect any CMD symptoms in the resistant genotype, nor did they prove the presence of the virus by PCR. The resistant genotype also had the best growth parameters and the highest cassava yield.

The experiments were well established and performed. The data obtained allow to achieve the stated objective of the work, which is to diagnose CMD using the "PlantVillage Nuru App" application and the laboratory diagnostic method of PCR and to compare the agronomic parameters of plants with different degrees of CMD expression.

Reviewer #3 Comments: The authors need to better evaluate the data obtained and draw proper conclusions from it, which according to the manuscript submitted, they have failed to do.

Response: Thanks for your inputs. This has been done.

Reviewer #3 Comments: In the discussion on lines 305-322, they correctly define the concepts of resistance and tolerance of plants to the pathogen and correctly state that the degree of resistance cannot be determined by symptoms alone, but that a diagnostic method must be used to detect symptomless infection.

Response: Thanks for the comments.

Reviewer #3 Comments: It is not appropriate to write on lines 323-324 that the SLICASS 4 genotype is potentially resistant when the results of the paper show that it is indeed resistant to the viruses causing CMD.

Response: The sentence has been recast to reflect the true picture of the result.

Reviewer #3 Comments: Then, on lines 333-339, it is written that plants based on symptomatic and symptomless plants of the susceptible variety are symptomless, i.e. tolerant to infection, when it is clear from the data presented that they had symptoms. It goes without saying that the intensity of symptoms differs in plants already infected at the time of planting from those that were infected from surrounding plants only during the course of the experiment. It is therefore by no means a question of tolerance.

Response: The corrections have been done accordingly.

Reviewer #3 Comments: Why do the authors write on line 364 that the application of Nuru was not effective in determining symptoms in symptomless plants when they already write on line 373 that the application was effective?

Response: The inconsistency has been resolved.

Reviewer #3 Comments: I miss the results of the CMD prevalence survey described in Section 2.1.

Response: During the survey, the Nuru App was utilized to detect the disease status of the planting materials into two categories including symptomatic and asymptomatic parental plants. Cuttings from these plants were used for the field experiment. Thus, data were not collected on CMD prevalence during the survey.

Reviewer #3 Comments: I recommend reworking the discussion and interpreting the data correctly.

Response: Thanks for your suggestion. Corrections have been done accordingly.

Further comments:

line 39: cocoa – if you mean the name of the variety, it should be capitalized. The same problem appeared on lines 182, 184, 186, 190, 191, 194, 195, 199, 200, 206, 215, 234, 247, 248, 260, 263, 297, 333, 339, 377

Response: Thanks for your suggestion. The first letter of the variety name has been capitalized as suggested.

Reviewer #3 Comments: line 129: Port Loko District in the Northern province - according to other parts of the text, it is the Northwestern province

Response: Thanks for your observation. Port Loko District is in the Northwestern province. The correction has been done (lines 130 and 141).

Reviewer #3 Comments: line 131: improved released genotype - indicate what the improvement in the genotype is - probably resistance to CMD; and continue to state it further in the text

Response: The information has been added as suggested (lines 144-147).

Reviewer #3 Comments: line 144: The plant height (cm), stem girth (mm), and number of nodes. - the sentence looks incomplete

Response: Thanks for your inputs. The corrections have been done accordingly as the incomplete sentence has been deleted since it was mistakenly added.

Reviewer #3 Comments: line 155: For each treatment in the trial - the word treatment is used incorrectly, it should be a variant of experiment; and continue to state it further in the text

Response: The correction has been done across the document and is highlighted red.

Reviewer #3 Comments: line 168: and an additional 10 min cycle at 72 °C. - the added 10 minutes cannot be called a cycle

Response: I agree with you. The correction has been done by deleting cycle.

line 171: 50 ng ultrapure water - this number is probably wrong

Response: The correction has been done and can be traced on line 188.

Comments on the Quality of English Language

Response: We have thoroughly read and revised the document.

Reviewer #3 Comments: Revising the English will help make the manuscript more readable.

Response: We have thoroughly read and revised the manuscript accordingly.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have corrected a number of factual errors and typos in the manuscript.

However, one key passage at the end of the discussion chapter (lines 357-361), where the authors misinterpret the degree of plant resistance to CMD, remains uncorrected.

They wrote this:

„Thus, the plants from cuttings of symptomatic and asymptomatic mother plant infected with ACMV and EACMV are healthy carriers (tolerant varieties) and could therefore constitute an important plant reservoir that contribute to spreading of the diseases. The tolerant plants from cuttings of asymptomatic Cocoa should not be used as a solution for the management and/or control of CMD.“

That's not true. The symptomatic and symptomless Cocoa plants from their experiment are not tolerant - they would have to be symptomless and with high virus concentrations throughout their lives, as the authors correctly describe on lines 331-337. The Cocoa plants they describe are in fact susceptible - they show symptoms of virus infection and spread the virus to their surroundings. The truth is that they cannot be used for planting in environments with high virus infection pressure.

 

Other errors that have not yet been corrected:

line 182: 50 ng ultrapure water - this number or units are wrong

 

I request that all listed errors be corrected before the manuscript is accepted for printing.

Author Response

Njala University (NU),

Njala Campus, Sierra Leone,

26^th April, 2025.

 

Dear Editor,

It is my pleasure to resubmit the revised version of the manuscript plants-3502534 entitled “Identification and Distribution of Begomoviruses Infecting Cassava Fields in Sierra Leone”. The manuscript has been revised as suggested by the Reviewer and corrections can be traced in red font where appropriate. The responses to the reviewer’s comments are below. We hope to hear from you about this manuscript soon.

 

Best regards,

Musa Decius Saffa

REVIEWER 3

Comments and Suggestions for Authors

Authors Musa Decius Saffa and co-workers presented here a manuscript entitled „Detection of Cassava Mosaic Disease and Assessment of Selected Agronomic Traits of Cassava (Manihot esculenta)“.

The authors used the "PlantVillage Nuru App" to detect viruses causing cassava mosaic disease in Sierra Leone, along with methods for virus indexing, evaluating the number of whiteflies on the crop, and assessing yield parameters of cassava with varying degrees of virus disease infection.

A survey of cassava mosaic disease (CMD) incidence was conducted on 80 farms in 4 regions of Sierra Leone. Field experiments were conducted in the same 4 regions. Cuttings were planted from symptomatic and symptomless plants of the susceptible variety and from the resistant variety. Highly susceptible infected plants were placed at the edges of the field as a source of virus.

CMD incidence, number of whiteflies on plants, plant growth and root yield were assessed.

In addition to phenotypic evaluation of symptoms, PCR was used to determine the presence of the virus.

The authors also evaluated the correlations between the different factors observed in the experiment.

The authors found the highest level of CMD symptoms in the variant of the experiment established from symptomatic plants. They did not detect any CMD symptoms in the resistant genotype, nor did they prove the presence of the virus by PCR. The resistant genotype also had the best growth parameters and the highest cassava yield.

The experiments were well established and performed. The data obtained allow to achieve the stated objective of the work, which is to diagnose CMD using the "PlantVillage Nuru App" application and the laboratory diagnostic method of PCR and to compare the agronomic parameters of plants with different degrees of CMD expression.

Reviewer #3 Comments: The authors need to better evaluate the data obtained and draw proper conclusions from it, which according to the manuscript submitted, they have failed to do.

Response: Thanks for your inputs. This has been done.

Reviewer #3 Comments: In the discussion on lines 305-322, they correctly define the concepts of resistance and tolerance of plants to the pathogen and correctly state that the degree of resistance cannot be determined by symptoms alone, but that a diagnostic method must be used to detect symptomless infection.

Response: Thanks for the comments.

Reviewer #3 Comments: It is not appropriate to write on lines 323-324 that the SLICASS 4 genotype is potentially resistant when the results of the paper show that it is indeed resistant to the viruses causing CMD.

Response: The sentence has been recast to reflect the true picture of the result.

Reviewer #3 Comments: Then, on lines 333-339, it is written that plants based on symptomatic and symptomless plants of the susceptible variety are symptomless, i.e. tolerant to infection, when it is clear from the data presented that they had symptoms. It goes without saying that the intensity of symptoms differs in plants already infected at the time of planting from those that were infected from surrounding plants only during the course of the experiment. It is therefore by no means a question of tolerance.

Response: The corrections have been done accordingly.

Reviewer #3 Comments: Why do the authors write on line 364 that the application of Nuru was not effective in determining symptoms in symptomless plants when they already write on line 373 that the application was effective?

Response: The inconsistency has been resolved.

Reviewer #3 Comments: I miss the results of the CMD prevalence survey described in Section 2.1.

Response: During the survey, the Nuru App was utilized to detect the disease status of the planting materials into two categories including symptomatic and asymptomatic parental plants. Cuttings from these plants were used for the field experiment. Thus, data were not collected on CMD prevalence during the survey.

Reviewer #3 Comments: I recommend reworking the discussion and interpreting the data correctly.

Response: Thanks for your suggestion. Corrections have been done accordingly.

Further comments:

line 39: cocoa – if you mean the name of the variety, it should be capitalized. The same problem appeared on lines 182, 184, 186, 190, 191, 194, 195, 199, 200, 206, 215, 234, 247, 248, 260, 263, 297, 333, 339, 377

Response: Thanks for your suggestion. The first letter of the variety name has been capitalized as suggested.

Reviewer #3 Comments: line 129: Port Loko District in the Northern province - according to other parts of the text, it is the Northwestern province

Response: Thanks for your observation. Port Loko District is in the Northwestern province. The correction has been done (lines 130 and 141).

Reviewer #3 Comments: line 131: improved released genotype - indicate what the improvement in the genotype is - probably resistance to CMD; and continue to state it further in the text

Response: The information has been added as suggested (lines 144-147).

Reviewer #3 Comments: line 144: The plant height (cm), stem girth (mm), and number of nodes. - the sentence looks incomplete

Response: Thanks for your inputs. The corrections have been done accordingly as the incomplete sentence has been deleted since it was mistakenly added.

Reviewer #3 Comments: line 155: For each treatment in the trial - the word treatment is used incorrectly, it should be a variant of experiment; and continue to state it further in the text

Response: The correction has been done across the document and is highlighted red.

Reviewer #3 Comments: line 168: and an additional 10 min cycle at 72 °C. - the added 10 minutes cannot be called a cycle

Response: I agree with you. The correction has been done by deleting cycle.

line 171: 50 ng ultrapure water - this number is probably wrong

Response: The correction has been done and can be traced on line 188.

Comments on the Quality of English Language

Response: We have thoroughly read and revised the document.

Reviewer #3 Comments: Revising the English will help make the manuscript more readable.

Response: We have thoroughly read and revised the manuscript accordingly.

Author Response File: Author Response.pdf