Loop-Mediated Isothermal Amplification Assay for Visual Detection of Salmonella enterica Serovar Typhimurium in Food Animal Meat Products
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript investigated the loop-mediated isothermal amplification assay for visual detection of Salmonella enterica serovar typhimurium in food animal meat products. The objectives are clear and the study has some significance. However, there are still some points that need to be revised.
- The layout of some tables needs to be optimized, and the alignment of some columns in Table 1 is inconsistent.
- The manuscript only mentions four gradients of betaine concentration optimization (1.0-1.5 M), but does not explain the basis for choosing this range. It is unclear whether it is based on preliminary experiments or literature.
- Centrifugal force should be italicized, such as line 111, line 115 and line 117, and so on.
- Line 89: Change “n=81” into “n = 81”. Numbers and symbols are not separated by spaces, and the same goes for the same line.
- Please check the all reference style and follow the guidelines of this journal.
- The positive rate of Typhimurium in 208 meat samples was as high as 89-90%, which is much higher than the 40%-60% data reported in the literature for the Philippine market. It is necessary to discuss whether the omission of the isolation step led to false positive results.
- In the sensitivity test, the detection limit of LAMP for pure culture is “4.98×10⁴ CFU/mL”, which is the same as that of PCR. However, the results of DNA dilution sensitivity and pure culture sensitivity show that LAMP is 100-fold more sensitive than PCR. Please explain the reasons for the difference between the DNA sensitivity and the bacterial culture sensitivity results.
- The study developed a closed-tube calcein-based LAMP detection method targeting the STM4497 gene of Typhimurium, which shows certain innovativeness. However, it fails to fully compare the advantages of this method with existing LAMP techniques, and relevant discussions need to be supplemented.
Author Response
Please see attachment. Thank you.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript "Loop-Mediated Isothermal Amplification Assay for Visual Detection of Salmonella enterica Serovar Typhimurium in Food Animal Meat Products" evaluated the application of a calcein-based and closed-tube loop-mediated isothermal amplification (LAMP)-based assay to detect S. Typhimurium in raw meat. Overall, the presentation is sound and reports interesting results to researchers in the subject. I have only minor considerations for the authors:
1) Section 3.1: Please specify in more detail the operational parameters tested to obtain the optimum PCR and LAMP protocols. Were both obtained from the literature? If so, was the system studied the same as in the current study?
2) Section 3.6: Since both PCR and LAMP have a high sensitivity in detecting Salmonella Typhimurium with similar LOD, how do the authors explain the differences in results between the PCR and LAMP assays? Which of the two, if there is one, could be providing more accurate results?
3) Please include a table comparing PCR and LAMP applications to your system, highlighting the advantages and drawbacks fairly.
4) Please also include a Table in the discussion section comparing the use of different genes to detect S. Typhimurium in LAMP-based assays.
Author Response
Please see attachment. Thank you.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsHere are my suggestions:
Major:
-Have authors compared the detection efficiency of cultivable methods and PCR-based methods? Please add this information.
- In Figure 2C, there are a lot of PCR products in the positive lanes. What is the reason for that? A single band of 200 bp is expected.
- The DNA dilution test showed a 100-fold higher sensitivity of the loop amplification test compared directly with PCR. However, both assays using DNA from colony-forming units showed similar sensitivity. Therefore, it is very categorical to state that the loop test is 100 times more sensitive than the traditional PCR assay. It depends on the sample; also, the purity of DNA molecules might diminish the sensitivity.
-The discussion section is too long. Please make it shorter.
Author Response
Please see attachment. Thank you.
Author Response File: Author Response.pdf
