Multiplexed Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Nucleic Acid-Based Lateral Flow Dipstick as a Rapid Diagnostic Method to Detect SARS-CoV-2
Round 1
Reviewer 1 Report
The paper discusses a topic of interest, that is the development of a rapid molecular diagnostic assay for the detection of SARS-CoV-2. The assay is based on LAMP amplification and rapid detection by lateral flow technique. The methodology of the study is accurate, the results are optimally presented, and the discussion takes into account the main evidence in the field. Strengths and limitations of the study are also reported. I suggest minor revisions to improve the quality of the manuscript before publication.
1. In materials and methods subsections 2.2, 2.3, 2.4, 2.5, 2.6 are duplicated. Please revise the text by removing the duplicated parts.
2. Lines 64-68: LAMP assays are commonly used in diagnostic microbiological field. The sentence should be expanded to include the application of LAMP rapid tests for the detection of nosocomial bacteria (e.g. Acinetobacter baumannii, doi: 10.1016/j.jhin.2021.09.015), fungal pathogens (e.g. Pneumocystis jirovecii doi: 10.1111/myc.13152) and antibiotic resistance genes (e.g. β-lactamases genes, doi: 10.1093/jac/dkac230).
3. Lines 200-2012: LFD Analyses is an essential part in the proposed LAMP protocol. This part should be described in more detail (principle of detection, preparation of lateral flow support and procedures).
4. The image quality of figures 1-5 could be improved.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Simon and co-author performed good research for the development of rapid LAMP detection of SARS-CoV2. The used methods are well-described and can be easily reproduced. Most of the conclusions are proved by experiments. The main drawbacks of the paper is presentation of the result and specificity tests. However, I have several remarks and questions:
1. Line 45-46: … antigen rapid tests (RTK-Ag), antibody rapid tests (RTK-Ab), cell culture, electron microscopy to the chest, and CT-scans [9]. However, these techniques are limited by the lack of facilities, trained personnel, and accuracy.
Disagree with the statement about RTK-Ag and RTK-Ab. These lateral flow-based tests are the simplest one and can be performed even in non-laboratory conditions. Often no viral RNA extraction or sample preparation requires for the tests.
2. lines 153 – 212 is a copy of lines 101-146
3. Fig 2:
please provide plot in coordinates DNA amount – Ct and move the curves (Fig 2A) in the Supplementary information
please align marks (e.g. (B), (D), (F)) in column. It would be easier for readers.
4. Fig. 3: see the comment to Fig 2
5. Fig. 4: see the comment to Fig 2
6. Please add in the manuscript amount of viral RNA or cDNA used in the specificity test
7. Lane 331: other coronaviruses produced negative results as shown by the absence of ladder-like band
In samples of IBV after LAMP kind of non-specific smear is present. Is some potential region in IBV genome for LAMP primers recognition? Perhaps the smear was caused by an excess of loaded genomic DNA? During lateral flow assay such unspecific products cause false-positive results. Did you check it? I propose to verify LAMP-LFD with IBV.
8. Did you evaluate the sensitivity of the LAMP-LFD? If yes, you should mention the results in the corresponding section. I do not insist on this experiment due to your argumentation in lines 418-424.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Reviewer 3 Report
Journal Microorganisms (ISSN 2076-2607)
Manuscript ID microorganisms-2373658
Type Article
Title. Multiplexed Reverse Transcription Loop-mediated Isothermal Amplification Coupled with a Nucleic Acid based Lateral Flow Dipstick as a Rapid Diagnostic Method to Detect SARS-CoV-2
Main comments.
1. Which exception (please see below) work is generally well written and the experimental design well conducted. A novel highly sensitive and rapid SARS-CoV-2-based loop-mediated isothermal amplification (LAMP) method was meticulously developed.
2. There are numerous repetitions in the methods section. It seems that 2.1 -2.7 sections are repeated with other numerations. Pease carefully revise the work in order to eliminate these errors.
3. The lack of validation of the method with clinical samples is a limitation of the study which should be underlined in the discussion
4. Line 14 “specificity and sensitivity that rivals the gold standard” I would mitigate this point. Specificity and sensitivity can only be computed by testing a large set of clinical samples previously screened with a gold standard method.
Minor comments:
1. Authors are encouraged to include and discuss the following works describing LAMP methods for detecting SARS-CoV-2:
a. https://virologyj.biomedcentral.com/articles/10.1186/s12985-020-01435-6
b. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8802757/
c. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9029081/
2. Lines 10-11”rates” repetition
3. More supporting references should be included in the methods
4. The most recently developed strategies for detecting SARS-CoV-2 have been extensively described in these reviews (PMID: 35744711, https://www.nature.com/articles/s41563-020-00906-z and PMID: 35709204). I Kindly suggest the authors to include these additional works in the introduction
5. Please check the repetitions throughout the manuscript. For instance PCR as been mentioned multiple times (lines 48/49-144) times with its complete name Polymerase Chain Reaction, while only acronyms can be used
6. Figure quality be improved. Numerous figures are unsymmetrical and words are very difficult to be read
7. Please remove space lines between paragraph in the discussion
8. 317 and 427 it should be SARS-CoV-1
In general well written, no particular comment
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
I accept all authors' responses. Thank you for performing experiments and updating the figures.
Reviewer 3 Report
The manuscript can be accepted in the present form
