Application of Loop-Mediated Isothermal Amplification Assay Combined with Lateral Flow Dipstick (LAMP-LFD) for Specific and Sensitive Detection of Acidovorax citrulli (Schaad et al.) Causing Bacterial Fruit Blotch in Cucurbit Plants
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript “Application of Loop-Mediated Isothermal Amplification Assay Combined with Lateral Flow Dipstick (LAMP-LFD) for Specific and Sensitive Detection of Acidovorax citrulli (Schaad et al.) Causing Bacterial Fruit Blotch in Cucurbit Plants” presents a new system for an on-site detection method for this important pathogen. The subject is well introduced, pointing out the damages and economic losses of this disease, and the different detection protocols that have been developed until now.
In general, the work addresses an important aspect in detection. The stages of the research are well designed and performed, and it seems that the final objective of the work have been accomplished. The primers and probe designed, strains analysed and the different analyses performed (optimization of the protocol, specificity and sensitivity assays, and plant material employed) are logical and well presented.
However, some questions arise in this work. The first one is the need of a lateral flow dipstick system to achieve the visualization of the analyses, when no increase in sensitivity is obtained. Why not using just the visual detection with the UV light, which offers a better visualization than the colour change, instead of the dipstick process? The outcome is the addition of several steps and the use of more molecular products that increase the time and money of the procedure.
Another aspect is the introduction and discussion sections. They describe the previous detection systems developed compared to the one proposed in this work, but in the end it is very repetitive, and no new information is provided, so the discussion should be rewritten.
The most important difficulty of this work is understanding the sensitivity results. First, the optimization assays conclude that at 30 min you have positive results, quite clear under UV light. But because of the use of the dipstick, you have to increase to 60 min to visualize in an agarose gel (but you are not using it, it was just as a matter of confirmation in this paper) plus the steps of adding the reagents to the tubes and the hybridization and reaction time (15 plus 10 more min) needing in total 85 min, and with steps that can contribute to contamination of the samples.
I don’t see the match between the values provided with the results presented in the figures. In fig. 6, the test bank starts with 10 µg.µL-1, but the following concentration is 1 pg, no 1 µg, while the rest are 10 times serial dilutions. In this figure, the results are coincident with the values obtained, but in fig. 7 there is no coincidence. The number of samples showed are 8, but in the text appear 12 (from 107 to 1 CFU/mL). Second, but more important, the picture shows a positive result reaching only 103 CFU/mL, not 1 CFU/mL as stated. Lanes 6 and 7 are negative in all detection systems employed (visual, UV and dipstick) which are 102 and 50 CFU/mL respectively. Which is the correct sensitivity of the assay?
Finally, editing comments: in pg 17, lines 544-547 it is stated that “The results of an examination of specificity indicated that the designed LAMP primers and probes only amplified A. citrulli DNA, with amplification signals observed with non-target test strains…” it should be “the designed LAMP primers and probes only amplified A. citrulli DNA, with NO amplification signals…”
In line 567 is stated “Cho et al. established an A. citrulli SYBR Green-based real-time quantitative fluorescent PCR technique with a detection sensitivity of 5 fg/μL for A. citrulli genomic DNA, which was equivalent to 6.5 CFU/mL –1. When the results of the LAMP are stated in this work, the sensitivity is 1 fg/µL, and 8 CFU/mL, while with 5 fg/μL you reach a lower value (6,5 CFU/mL) instead, why this different correspondence?.
In line 584: “The sensitivity of our assay was comparable to that of the A. citrulli locked nucleic acid real-time PCR assay established by Nurhan and Hüseyin [73] but lower than that of the A. citrulli ddPCR [61], LAMP [33] and SYBR Green-based real-time fluorescent quantitative PCR [38] assays established by Lu et al., Yan et al., and Cho et al., respectively”. Lower here means that the sensitivity is not as good as the ones compared to, and the authors probably want to stress that the LAMP-LFD is better, so the sensitivity is higher, no lower…
Then, with these inconsistencies and contradictions in the results a revision in these aspects must be solved before being accepted for publication. Also, the vision of using a LFD instead of the LAMP for the visual detection should have to be well explained, I don’t see any advantage in choosing this option.
Author Response
Reviewer 1
Comments 1: The manuscript “Application of Loop-Mediated Isothermal Amplification Assay Combined with Lateral Flow Dipstick (LAMP-LFD) for Specific and Sensitive Detection of Acidovorax citrulli (Schaad et al.) Causing Bacterial Fruit Blotch in Cucurbit Plants” presents a new system for an on-site detection method for this important pathogen. The subject is well introduced, pointing out the damages and economic losses of this disease, and the different detection protocols that have been developed until now.
Response 1: Thank you for review, we agree with this comment.
Comments 2: In general, the work addresses an important aspect in detection. The stages of the research are well designed and performed, and it seems that the final objective of the work has been accomplished. The primers and probe designed, strains analyzed and the different analyses performed (optimization of the protocol, specificity and sensitivity assays, and plant material employed) are logical and well presented.
Response 2: Thank you for review, we agree with this comment.
Comments 3: However, some questions arise in this work. The first one is the need of a lateral flow dipstick system to achieve the visualization of the analyses, when no increase in sensitivity is obtained. Why not using just the visual detection with the UV light, which offers a better visualization than the color change, instead of the dipstick process? The outcome is the addition of several steps and the use of more molecular products that increase the time and money of the procedure.
Response 3:
Thank you for pointing this out. We agree with this comment. Researchers often use liquid color change or ultraviolet observation of white precipitate methods to determine LAMP amplification products. Although these methods are simple and visual, they have the following disadvantages: (i) LAMP detection methods involve the design of primers. If the quality of the designed primers is not ideal, dimers are easily generated during LAMP amplification. When the concentration of dimers reaches a certain amount, the detection results based on color change or ultraviolet methods are positive, which can easily lead to false positives and incorrect detection results; (ii) Using nucleic acid dyes to induce color changes in LAMP amplification products can easily lead to aerosol deposition and false positives.
LFD (lateral flow dipstick) is an addition of a probe that can specifically hybridize with LAMP products on the basis of LAMP method. Adding a probe is equivalent to adding a specific detection site, improving the detection specificity. LFD has the following advantages: LFD probes (HP) are designed based on conserved sites in the target gene sequence of the detection pathogen, highly matching the sequence of the corresponding site of the target gene. Positive reactions can only occur when the probe (HP) specifically hybridizes with LAMP products. The combination and confirmation of four LMAP amplification product detection methods, including color change, ultraviolet white precipitation observation, 2% agarose gel electrophoresis and LFD, effectively overcome the false positive defect caused by aerosol deposition and dimer, and further improve the specificity and accuracy of LAMP detection method.
Comments 4: Another aspect is the introduction and discussion sections. They describe the previous detection systems developed compared to the one proposed in this work, but in the end it is very repetitive, and no new information is provided, so the discussion should be rewritten.
Response 4:
We agree with this comment. We have adopted the suggestions of the reviewers and rewritten some of the content in the discussion section. Added the following content: (1) Why add LFD method, what advantages does LFD have, and it can solve what problems. (2) When LAMP-LFD was used to detect A. citrulli, its detection efficiency for genomic DNA was superior to Cho et al.'s method, but its detection sensitivity for A. citrulli bacterial suspension was lower than Cho et al.'s method. What is the reason for this result? (3) How to improve the accuracy of LAMP-LFD when testing seeds with low infection level.
Comments 5: The most important difficulty of this work is understanding the sensitivity results. First, the optimization assays conclude that at 30 min you have positive results, quite clear under UV light. But because of the use of the dipstick, you have to increase to 60 min to visualize in an agarose gel (but you are not using it, it was just as a matter of confirmation in this paper) plus the steps of adding the reagents to the tubes and the hybridization and reaction time (15 plus 10 more min) needing in total 85 min, and with steps that can contribute to contamination of the samples.
Response 5: Agree, the most important difficulty in this study is understanding the sensitivity results. Yes, when the reaction time is 30 minutes, we can get the positive result through color change and ultraviolet observation, but we can't judge whether the amplification product is the best. So we use 2% agarose gel electrophoresis to detect the amplification product, and judge the amplification product by the brightness of the electrophoresis band. The brightest band indicates that the amplification product is the most, and the amplification reaction efficiency is the best under this condition.
After 30 minutes of reaction, positive results can also be obtained by 2% agarose gel electrophoresis, but it is not the best result because its bands are not the brightest. When using 2% agarose gel electrophoresis to detect the amplification product, no reagent was added to the product, and no dipstick was used. It was not because of the use of dipstick that the reaction time was increased to 60 minutes.
Adding other reaction buffers and probes to the amplification product is a hybridization step to obtain the hybridization product for detection using LFD dipstick. LFD dipstick is used to verify the accuracy of the other three methods (color change, ultraviolet white precipitation observation and 2% agarose gel electrophoresis).
We fully agree with your comment that adding reagents to the test tube should cause sample contamination. However, after using a hybridization probe, we were able to effectively eliminate the impact of contamination on the results. This is because the probe is designed based on gene target gene specific sites and has strong specificity for the amplification product. Only when the probe can specifically hybridize with the amplification product, the hybrid products can be produced, the results of LFD dipstick is positive.
Comments 6: I don’t see the match between the values provided with the results presented in the figures. In fig. 6, the test bank starts with 10 µg.µL-1, but the following concentration is 1 pg, no 1 µg, while the rest are 10 times serial dilutions. In this figure, the results are coincident with the values obtained, but in fig. 7 there is no coincidence. The number of samples showed are 8, but in the text appear 12 (from 107 to 1 CFU/mL). Second, but more important, the picture shows a positive result reaching only 103 CFU/mL, not 1 CFU/mL as stated. Lanes 6 and 7 are negative in all detection systems employed (visual, UV and dipstick) which are 102 and 50 CFU/mL respectively. Which is the correct sensitivity of the assay?
Response 6: Thank you for pointing out this out. We have carefully checked and modified all the data in the figures, tables, and the entire text. The corrected data no longer contains any contradictions or discrepancies. Thank you again for your careful and meticulous review.
Comments 7: Finally, editing comments: in pg 17, lines 544-547 it is stated that “The results of an examination of specificity indicated that the designed LAMP primers and probes only amplified A.citrulli DNA, with amplification signals observed with non-target test strains…” it should be “the designed LAMP primers and probes only amplified A. citrulli DNA, with NO amplification signals…”
Response 7: We agree with this comment. “The results of an examination of specificity indicated that the designed LAMP primers and probes only amplified A.citrulli DNA, with amplification signals observed with non-target test strains…” has been corrected by “The results of an examination of specificity indicated that the designed LAMP primers and probes only amplified A. citrulli DNA, with No amplification signals observed in non-target strains…” in revised manuscript.
Comments 8: In line 567 is stated “Cho et al. established an A.citrulli SYBR Green-based real-time quantitative fluorescent PCR technique with a detection sensitivity of 5 fg/μL for A.citrulli genomic DNA, which was equivalent to 6.5 CFU/mL. When the results of the LAMP are stated in this work, the sensitivity is 1 fg/µL, and 8 CFU/mL, while with 5 fg/μL you reach a lower value (6.5 CFU/mL) instead, why this different correspondence?.
Response 8: Thank you for pointing out this out. Why does this phenomenon occur? The author explains as follows: there are differences in DNA concentration and quality obtained by different extraction methods for bacterial genomic DNA.
The sensitivity of the A.citrulli LAMP-LFD assay in detection A.citrulli genomic DNA is 1 fg.µL-1, while the SYBR Green based real-time quantitative fluorescent PCR technique established by Cho et al. is 5 fg.μL-1, which indicated the sensitivity of the A.citrulli LAMP-LFD assay is higher than that of the method established by Cho et al.. In the sensitivity test of detecting bacterial suspension, the genomic DNA extracted from bacterial suspension of 6.5 CFU.mL-1 was 5 fg.μL-1 using method of Cho et al.. However, the genomic DNA from bacterial suspension of 8 CFU.mL-1 was only 1.0 fg.μL-1 in this study. Obviously, the bacterial genomic DNA extraction method adopted by Cho et al. is more effective than the method used in this study.
In the discussion section, the importance of bacterial genomic DNA extraction methods in improving detection sensitivity was discussed (see the revised manuscript discussion section).
Comments 9: In line 584: “The sensitivity of our assay was comparable to that of the A. citrulli locked nucleic acid real-time PCR assay established by Nurhan and Hüseyin [73] but lower than that of the A. citrulli ddPCR [61], LAMP [33] and SYBR Green-based real-time fluorescent quantitative PCR [38] assays established by Lu et al., Yan et al., and Cho et al., respectively”. Lower here means that the sensitivity is not as good as the ones compared to, and the authors probably want to stress that the LAMP-LFD is better, so the sensitivity is higher, no lower…
Response 9: Thank you for pointing this out. We agree with this comment. In order to stress that the sensitivity of the LAMP-LFD is better, we deleted the follow sentence (“The sensitivity of our assay was comparable to that of the A. citrulli locked nucleic acid real-time PCR assay established by Nurhan and Hüseyin [73] but lower than that of the A. citrulli ddPCR [61], LAMP [33] and SYBR Green-based real-time fluorescent quantitative PCR [38] assays established by Lu et al., Yan et al., and Cho et al., respectively”), and proposed some suggestions for further improving the detection sensitivity.
The sensitivity of pathogen detection is not only closely related to the detection method, reaction temperature, and reaction time, but also influenced by factors such as the selection of target gene, reagent concentration, primers and probe sequences [49, 71, 74]. Therefore, in order to further improve the sensitivity of the A. citrulli LAMP-LFD assay, it is necessary to re-optimize and combine the above-mentioned factors in future research.
The relevant modifications have been showed in page 16 (or Line 582-591) of the revised manuscript.
Comments 10: Then, with these inconsistencies and contradictions in the results a revision in these aspects must be solved before being accepted for publication. Also, the vision of using a LFD instead of the LAMP for the visual detection should have to be well explained, I don’t see any advantage in choosing this option.
Response 10: Thank you for pointing this out. The errors, inconsistencies and contradictions have been corrected in the revised manuscript.
Why use LFD to detect LAMP amplification products? Here is an explanation and clarification: Compared with using colorimetric method or ultraviolet methods to detect LAMP products, LFD has the following advantages: LFD probe (HP) is designed based on conserved sites in the target gene sequence of the detection object (A. citrulli), highly matching the sequence of the corresponding site of the target gene. Only probe (HP) specific hybridization with LAMP products can produce positive reactions, thus improving the specificity and accuracy of detecting LAMP amplification products.
LFD overcomes the following shortcomings of the LAMP amplification products detection method: 1. The LAMP detection method involves the design of primers. If the quality of the designed primers is not ideal, dimers are easily generated during LAMP amplification. However, based on color changes or UV methods, the detection results of primer dimers in nucleic acid amplification are positive, which can lead to false positives and incorrect detection results; 2. Usually, the LAMP amplification product is judged by adding DNA-intercalating dyes to trigger color change or observing the white precipitate under ultraviolet light, which can easily cause aerosol deposition or primer dimer problems, often leading to false positives and erroneous results. The use of LFD method precisely overcomes the above-mentioned false positive problem.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis is a carefully done study and the findings are of considerable interest. A few minor revisions are listed below.
Table 1: ‘Acidovorax citrulli’ should be Italic.
Table 1: ‘Citrullus lanatus’ should be Italic.
Table 2: A. citrulli in the table title should be Italic.
Figure 1: The underline of F3 only covers 15 bp. This does not match Table 3.
The red line indicating the HP primer only covers 17 bp. This red line only partially covers the 5' terminal g.
Line 311, 317, 383, 407: A. citrulli should be Italic.
Line 339: ‘Tiangen Biotech Co., Ltd.’ should move to line 171.
Line 378, 500: Acidovorax citrulli should read A. citrulli in Italic.
Line 362, 363, 374: There are some commas with different fonts.
Author Response
Reviewer 2
Comments 1: Table 1: ‘Acidovorax citrulli’ should be Italic.
Response 1: Agree. We have corrected it.
Comments 2: Table 1: ‘Citrullus lanatus’ should be Italic.
Response 2: Agree. We have corrected it.
Comments 3: Table 2: A. citrulli in the table title should be Italic.
Response 3: Agree. We have corrected it.
Comments 4: Figure 1: The underline of F3 only covers 15 bp. This does not match Table 3. The red line indicating the HP primer only covers 17 bp. This red line only partially covers the 5' terminal g.
Response 4: Thank you for pointing this out. We have redesigned the Figure 1 and extended the underline of F3 in the revised version. The extended underline covered all 17 letters (bp) of the F3. Using the same method, we extended the red line of HP. Extended red line covered all 18 letters (bp) of the HP.
Comments 5: Line 311, 317, 383, 407: A. citrulli should be Italic.
Response 5: Agree. We have corrected it.
Comments 6: Line 330: ‘Tiangen Biotech Co., Ltd.’ should move to line 171.
Response 6: Thank you for pointing this out. “Tiangen Biotech Co., Ltd.” has been moved to line 171.
Comments 7: Line 378, 500: Acidovorax citrulli should read A. citrulli in Italic.
Response 7: Thank you for pointing this out. “Acidovorax citrulli” has been replaced by “A. citrulli”
Comments 8: Line 362, 363, 374: There are some commas with different fonts.
Response 8: Thank you for pointing this out. The commas in the Line 362, 363 and 374 have been corrected in the some font (Italic).
Author Response File: 
Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsIn this study, the authors demonstrate LAMP-LFD method to detect the typical seed-borne bacterium Acidovorax citrulli. They confirmed the detection limit for pure genomic DNA, bacterial suspension, bacterial amounts on seed, and infection rate of seeds. In addition, they checked the detection specificity. My comments are as follows:
1. There are many times the authors mentioned the advantages of this method, however, each time they gave the results not totally the same, it would be better for the authors to give the readers a clear advantage for this method. Using LAMP to detect Acidovorax citrulli was reported many years ago, but the authors did not mention it in this manuscript.
2. Fig. 1 and Table 3 showed the same thing, it’s better to remove one.
3. Why did the authors choose Aave_2049 to design the primers? I asked because Aave_2049 is an rhs gene and there are many homologous genes in this bacterium.
4. In the Results, the authors sometimes did not cite the figure or table when showing the results.
5. There is something wrong in the figure legend of Fig.7.
6. “When the infection level of watermelon seeds was 0.05%–0.1%, the detection rate by LAMP-LFD was 20%–100%. Thus, the target pathogen can be detected in seed samples with 0.05% infection and above” I don’t think this conclusion is right.
7. Line 383 “1-3: Genomic DNA of A. citrulli”, are these samples totally the same? With the same concentration?
8. According to my knowledge, the “naturally infected” seeds should be positive, they are not equal to “commercially available”, however, in Fig. 8, 3 of 5 samples were detected as negative. The authors need to give some explanation.
9. The authors sometimes use Acidovorax citrulli Schaad et al. to represent this bacterium and sometimes Acidovorax citrulli, and many times they did not italicize.
10. It would be better to change the “rpm” to “g” when describing the centrifugation conditions.
11. As far as I know, there is no “、” in English writing.
Author Response
Reviewer 3
Comments 1: There are many times the authors mentioned the advantages of this method, however, each time they gave the results not totally the same, it would be better for the authors to give the readers a clear advantage for this method. Using LAMP to detect Acidovorax citrulli was reported many years ago, but the authors did not mention it in this manuscript.
Response 1: We agree with this comment. Therefore, according to the suggestions of the reviewers, we have made a comprehensive summary of its advantages (This assay thus offers the advantages, including easy operation, rapidity, high specificity and sensitivity, low cost (no need for complex and expensive precision instruments), visualization of detection results, good stability, and strong applicability.) in Line 34-35, Line 124-126, Line 612-614 and Line 624-625 in revised manuscript.
Yes, the LAMP method has been used to detect Acidovorax citrulli many years ago. The author mentioned this method in the discussion section (Line 563-565). Meanwhile, the detection sensitivity of this method was compared and analyzed with conventional PCR methods.
Comments 2: Fig. 1 and Table 3 showed the same thing, it’s better to remove one.
Response 2: The reviewer's suggestion is very good. But after careful consideration, we have decided to keep both of them, which will be more conducive to readers' reading and understanding.
Comments 3: Why did the authors choose Aave_2049 to design the primers? I asked because Aave_2049 is an rhs gene and there are many homologous genes in this bacterium.
Response 3: Thank you for pointing this out. We agree with this comment. The YD-repeat protein is one of the rhs genes, is widely present in many bacteria，and has a certain degree of conservation in different bacterial species. Primers and probes designed based on conserved sites (specific sites) of the YD-repeat protein gene of A. citrulli have high specificity for A. citrulli, such ensuring the accuracy of the results in detecting A. citrulli.
Comments 4：In the Results, the authors sometimes did not cite the figure or table when showing the results.
Response 4: Thank you for pointing this out. We agree with this comment. According to your findings, the figures and tables have been cited in revised manuscript when showing the results in section of results.
Comments 5：There is something wrong in the figure legend of Fig.7.
Response 5: Thank you for pointing this out. Something wrong in the figure legend of Fig.7 has been corrected in the revised manuscript.
Comments 6：“When the infection level of watermelon seeds was 0.05%–0.1%, the detection rate by LAMP-LFD was 20%–100%. Thus, the target pathogen can be detected in seed samples with 0.05% infection and above” I don’t think this conclusion is right.
Response 6: We agree with this comment. Yes, the conclusion (Thus, the target pathogen can be detected in seed samples with 0.05% infection and above) is not scientific enough. We have revised the conclusion as follows: "The higher the infection level of seeds, the higher the positive detection rate”. “To ensure the accuracy of the detection results, the sample size and number of replicates should be increased as much as possible in detecting A. citrulli”. See revised manuscript.
Comments 7: Line 383 “1-3: Genomic DNA of A. citrulli”, are these samples totally the same? With the same concentration?
Response 7: These genomic DNA were derived from different isolate of A. citrulli. The genomic DNA concentrations were not measured or regulated in the study. In theory, their concentrations were different.
Comments 8: According to my knowledge, the “naturally infected” seeds should be positive, they are not equal to “commercially available”, however, in Fig. 8, 3 of 5 samples were detected as negative. The authors need to give some explanation.
Response 8: We agree with this comment. The “naturally infected” seeds are not equal to “commercially available” seeds. In this study, we actually used "commercially available seeds" as samples, and not “naturally infected seeds”. We misunderstood and mistakenly used "commercially available seeds" as "naturally infected seeds". We have checked and corrected the entire text, and have written it as "commercially available seeds ".
Comments 9: The authors sometimes use Acidovorax citrulli Schaad et al. to represent this bacterium and sometimes Acidovorax citrulli, and many times they did not italicize.
Response 9: Thank you for pointing this out. The author has checked the correct scientific name, abbreviation, and italicization of "Acidovorax citrulli" throughout the manuscript. When "Acidovorax citrulli" first appears in the manuscript, its full scientific name is "Acidovorax citrulli Schaad et al.", and when it appears in the future, it is abbreviated as "A. citrulli" in italicization.
Comments 10: It would be better to change the “rpm” to “g” when describing the centrifugation conditions.
Response 10: Thank you for pointing this out. We agree with this comment. Therefore, the “rpm” has been changed to “g” when describing the centrifugation conditions.
Comments 11: As far as I know, there is no “、” in English writing.
Response 11: Thank you for pointing this out. “、” has been replaced by “,” in revised manuscript.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsAll the issues and comments reported have been revised and changed properly.
Only two small issues:
In pg. line 423, the sentence "The results were showed that when the bacterial count was..." should be "The results showed that when the bacterial count was..."
In pg 15, line 515 says: "we successfully designed and synthesized primers and probes specific..." I assume the authors do not synthesize the primers, but the company, that part should be deleted.
Author Response
Comments 1: In pg 12, line 423, the sentence "The results were showed that when the bacterial count was..." should be "The results showed that when the bacterial count was...".
Response 1: Thank you for review, we agree with this comment. The sentence" The results were showed that when the bacterial count was..." has been corrected as follow: "The results showed that when the bacterial count was...
Comments 2: In pg 15, line 515 says: "we successfully designed and synthesized primers and probes specific..." I assume the authors do not synthesize the primers, but the company, that part should be deleted.
Response 2: Thank you for pointing this out. We agree with this comment. , we agree with this comment. Yes, the specific primers and probes were designed by us, but they were synthesized by biotechnology companies. So, we change sentence "we successfully designed and synthesized primers and probes specific to A. citrulli …." to sentence "we successfully designed primers and probes specific to A. citrulli ….". “synthesized” has been deleted in revised manuscript.
Reviewer 3 Report
Comments and Suggestions for AuthorsNA
Author Response
Thank you for reviewing the manuscript. We have strictly followed your suggestions and made revisions to the manuscript. We have rewritten the introduction, result, especially the discussion and conclusion sections.
I hope our revised manuscript can be approved by you. Thank you again for your careful review.
The YD-repeat protein (Aave_2049) is one of the rhs genes, and there are many homologous genes among bacteria in the family Enterobacteriaceae. There is a certain degree of conservation in the YD-repeat protein gene sequences between different species. Therefore, real-time fluorescence quantitative PCR using primers and probes designed based on YD repeat protein gene conserved sites has been successfully applied in detection of A. citrulli.
