Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.
Round 1
Reviewer 1 Report
The multiplex RT PCR assay developed for five lily viruses is specific and sensitive and validated at field level, shows the potential for field monitoring and quarantine purpose for these viruses. The study is well planted and interpreted with suitable justifications. However, in material and methods under design of primers and TaqMan probes -the accession number of the complete genome sequences used in this study may also be provided.
Line no 200-202, should be check and corrected in accordance to results presented in Figure 8B.
Author Response
Dear reviewer:
We appreciate your valuable comments concerning our manuscript, which were helpful in improving the quality of our present study. Below you will find our point-by-point responses to your comments, which are marked in red in the paper.
1. In material and methods under design of primers and TaqMan probes -the accession number of the complete genome sequences used in this study may also be provided.
Response：Thanks for your very thoughtful suggestion. we provided the accession number of the complete genome sequences used to design primers and TaqMan probes （page 2, lines 86-92）.
2. Line no 200-202, should be check and corrected in accordance to results presented in Figure 8B.
Response：We have made corrections according to your good suggestion. In line 246, 51 samples co-infected with three viruses was replaced by 51 samples co-infected with four viruses
Reviewer 2 Report
Dear authors,
it is interesting paper about a rapid and sensitive procedure, based on the TaqMan real-time PCR assay, for the simultaneous detection and specific quantification of viruses in Lilium spp. But I have some concerns that prevent me to endorse its acceptance at the present stage.
Since you would like to publish your work in Agronomy, you might add short description of the studied viruses, with e.g. some data and information of losses of Lilium spp. (or other important plant species/crops) production caused by these particular viruses. These information should be part of the Introduction. Although known, but the abbreviatons in this section should be explained and very briefly the principle of the listed detection methods should be provided.
In the Materials and Methods section brief description of lily smaples used in this study need to be presented.
Check the spelling, there are few errors (e.g. „Fresh leaves from lily samples, which were confirmed to be infected with LSV, LMoV,58 CMV, SYSV, and PlAMV......).
Author Response
Dear reviewer,
We appreciate your valuable comments concerning our manuscript, which were helpful in improving the quality of our present study. Below you will find our point-by-point responses to your comments, which are marked in red in the paper.
1.Since you would like to publish your work in Agronomy, you might add short description of the studied viruses, with e.g. some data and information of losses of Lilium spp. (or other important plant species/crops) production caused by these particular viruses. These information should be part of the Introduction.
Response: Thanks for your very thoughtful suggestion. Description of the studied viruses was added in line 32-45.
2. Although known, but the abbreviatons in this section should be explained and very briefly the principle of the listed detection methods should be provided.
Response: Thanks for your very thoughtful suggestion. In line 50-54, abbreviatons were explained and principle of detection methods was provided briefly.
3. In the Materials and Methods section brief description of lily smaples used in this study need to be presented.
Response: Thanks for your good suggestion. Brief description of lily smaples used in this study were presented in line 71-72 and 156-157.
4. Check the spelling, there are few errors (e.g. „Fresh leaves from lily samples, which were confirmed to be infected with LSV, LMoV, CMV, SYSV, and PlAMV......).
Response: Thanks for your good suggestion. We checked the spelling and marked in red in the paper.
Reviewer 3 Report
This article focuses more on the methodology than conventional research. That is why 90% of the references are found in the introduction.
Although the authors provided convincing evidence of the efficiency and specificity of the method they presented, they did not give details on how they performed reverse transcription (RT) when they tested the samples collected during the field survey. I assume that the authors used 2 steps RT-Taq real-time PCR. However, they did not say anything about the multiplex RT. Did they prepare reactions mixtures with all the reverse primers together and in which volumes/concentrations or did they opt for something else? This detail has to be disclosed for reproducibility.
I will not be able to reproduce that experiment without that critical detail. This is the reason why I insist that the authors include that information in detail in the manuscript.
Author Response
Dear reviewer,
We appreciate your valuable comments concerning our manuscript, which were helpful in improving the quality of our present study. Below you will find our point-by-point responses to your comments, which are marked in red in the paper.
1. Although the authors provided convincing evidence of the efficiency and specificity of the method they presented, they did not give details on how they performed reverse transcription (RT) when they tested the samples collected during the field survey. I assume that the authors used 2 steps RT-Taq real-time PCR. However, they did not say anything about the multiplex RT. Did they prepare reactions mixtures with all the reverse primers together and in which volumes/concentrations or did they opt for something else? This detail has to be disclosed for reproducibility.
Response: Thanks for your very thoughtful suggestion. Yes, we used 2 steps RT-Taq real-time PCR. The details of RT and multiplex TaqMan real-time PCR sssay were provided in line 77-85 and 159-166. In this study, only oligo(dT)20 primer was used to prepare reverse transcription reaction mixtures.
Reviewer 4 Report
The paper entitled ,,Application of Multiplex TaqMan real-time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.” written by Leifeng Xu, Meng Song , Jun Ming and submitted to ,,Agronomy” under ID agronomy-1454563 describes development of mqRT-PCR assay for simultaneous detection of five viruses infecting lilies. The method can be useful as a fast and efficient diagnostic tool. The paper can be accepted after minor revision. Please find my specific comments below:
Line 32 - please add taxonomic information eg. genus, family
Line 57 – please add information about origin of the infected samples
Line 62 – what type of primers were used for reverse transcription?
Line 112-113 - please add taxonomic information eg. genus, family
Line 129 – when the samples were collected, what type of symptoms were observed?
Author Response
Dear reviewer,
We appreciate your valuable comments concerning our manuscript, which were helpful in improving the quality of our present study. Below you will find our point-by-point responses to your comments, which are marked in red in the paper.
1. Line 32 - please add taxonomic information eg. genus, family
Response: Thanks for your very thoughtful suggestion. In line 32-37, taxonomic information eg. genus, family of viruses were added.
2. Line 57 – please add information about origin of the infected samples
Response: The infected samples were collected from greenhouses at the Chinese Academy of Agricultural Sciences (Beijing, China) (line 71-72).
3. Line 62 – what type of primers were used for reverse transcription?
Response: oligo(dT)20 was used for reverse transcription (line 80).
4. Line 112-113 - please add taxonomic information eg. genus, family
Response: Taxonomic information eg. genus, family of SLRSV and ArMV was added in line 137-138.
5. Line 129 – when the samples were collected, what type of symptoms were observed?
Response: Samples with the symptoms of leaf mottle, leaf mosaic, leaf curling, chlorotic and yellow streaking, or flower color breaking were collected (line 156-157)
