WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva

David Ng; Ana Pinharanda; Merly C. Vogt; Ashok Litwin-Kumar; Kyle Stearns; Urvashi Thopte; Enrico Cannavo; Armen Enikolopov; Felix Fiederling; Stylianos Kosmidis; Barbara Noro; Ines Rodrigues-Vaz; Hani Shayya; Peter Andolfatto; Darcy S. Peterka; Tanya Tabachnik; Jeanine D’Armiento; Monica Goldklang; Andres Bendesky

doi:10.1371/journal.pone.0257464

Peer Review History

Original SubmissionJuly 21, 2021
4 Aug 2021 Decision Letter - Etsuro Ito, Editor PONE-D-21-23691 WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva PLOS ONE Dear Dr. Bendesky, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please revise the point about statistics. Please submit your revised manuscript by Sep 16 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The WHotLAMP manuscript from Ng et al presents a very good colorimetric LAMP assay for SARS-CoV-2 detection. There have been quite a few of these published now but the performance, simplicity, and novelty in this approach justify publication. The manuscript is clear and well written, and I feel it can be accepted mostly as is after addressing a few comments listed below. 1) The authors use an Orf1 primer set, it would be good to note whether the targeted region is anywhere close to the SGF deletion found in alpha, beta, gamma, and lambda variants which may affect performance. Related, the LOD is measured using quantitative PCR with N gene primers, a comment on the veracity of the assumption that 1 N=1 Orf1 would be good. 2) The authors describe spiking naked control RNA into saliva and then using it for detection. Other studies and conventional wisdom would say this wouldn't work and that the RNA is degraded extremely rapidly. Is there a reason it works here w/o RNase inactivation? 3) In describing the LoD the authors say that their assay is ">50X more sensitive than other recent saliva LAMP assays" which is a bit unfair considering this is contrived samples, data in Fig. 5A doesn't really indicate a "50X" sensitivity improvement. Plus there are a lot of LAMP assays, picking 1 for LOD comparison doesn't quite justify a claim of superiority to "other assays". Reviewer #2: Ng et al. have performed an interesting and clear study that may benefit the fight against the Covid-19 pandemic. The paper is clearly written and details a novel test for detecting the covid-19 spike protein in what appears to be a specific assay. The benefit of the assay is how easy it is to handle and that it can be translated to something that can be done at home, which allows more people to access a safe and specific covid-19 assay. Major comments: After reading the manuscript through and through several times, I can only make out one statistical test, however what test this was used is not mentioned. The Paper would greatly benefit from adding statistics throughout the paper and the statistics should be described in detail in the material and methods. It is not clear how many samples that has been analyzed, there is a mention of 20/20 positive samples and then 21/21. A table of the patient/sample characteristics detailing the number of positive samples, the severity of disease, negative controls and other diseases would greatly benefit the paper. ****** 6. 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Revision 1
31 Aug 2021 Author Response We appreciate the thoughtful reviews and the opportunity to respond to the Reviewers’ concerns and suggestions. As detailed below, we have addressed all points by adding new data and statistical tests, new analyses and figures, and rewriting sections of the manuscript to improve clarity. Reviewer 1: 1) The authors use an Orf1 primer set, it would be good to note whether the targeted region is anywhere close to the SGF deletion found in alpha, beta, gamma, and lambda variants which may affect performance. We have added a new figure panel (Figure 2B) that shows how our LAMP amplicon does not overlap any mutations in the alpha, beta, gamma, nor delta variants, suggesting our assay should be able to detect all these SARS-CoV-2 variants. Related, the LOD is measured using quantitative PCR with N gene primers, a comment on the veracity of the assumption that 1 N=1 Orf1 would be good. While SARS-CoV-2 virions package a single RNA genome, it is true that different SARS-CoV-2 genes are present at different levels in infected cells (Kim et al., 2020). In Figure 1B, we quantified the sensitivity of our assay using contrived saliva samples spiked with synthetic virions each containing single RNA viral copies (so each viral gene is 1 copy) and using LAMP primers targeting the N and E genes. This showed that we could detect as few as 4 viral particles per microliter of saliva (Fig 1A). As a more realistic measure of the limit of detection of our assay, we quantified the number of N gene RNA copies in saliva from COVID-19 patients through qPCR using the CDC approved N gene primers. This, followed by serial dilutions of these saliva samples showed that our LAMP assay has a limit of detection of ~3.6 N gene RNA copies per microliter of saliva. We have rewritten the text so it is clear that our limit of detection in saliva with people with COVID-19 is measured in terms of RNA copies of the N gene. 2) The authors describe spiking naked control RNA into saliva and then using it for detection. Other studies and conventional wisdom would say this wouldn't work and that the RNA is degraded extremely rapidly. Is there a reason it works here w/o RNase inactivation? We agree with the reviewer that because of the high level of RNases present in saliva, direct spike-in of naked RNA into saliva would lead to rapid degradation, as we (data not shown) and others have observed. In this assay (Figure 1A in the original manuscript), our goal was to determine whether Whatman paper had the potential to capture SARS-CoV-2 RNA in saliva. We would like to point out to that in this assay, saliva was initially mixed and pre-incubated with a lysis buffer with a final concentration of 0.4 M guanidine hydrochloride (GH) before the addition of naked RNA. Although the concentration of GH is insufficient to denature and fully inactivate RNases, it has been reported that low concentrations of GH can partially inactivate RNaseA very rapidly. For example at a concentration of 0.5 M GH, about 50% of RNaseA activity is lost (Liu and Tsou, 1987). While the precise species of RNase(s) in the saliva samples is not known, this study supports the notion that pre-treatment with GH inhibits some ribonuclease activity. We suggest that under the conditions used in this assay, there is partial inactivation of RNases in the saliva sample during the pre-incubation step. Combined with the high number (100,000) of RNA copies added and brief incubation time with Whatman paper (~1 min) before washing, sufficient RNA target is bound to the filter paper to be detected using LAMP. Because RNases are likely only partially inactivated under these conditions, we suspect that longer exposure times of naked RNA to saliva will lead to the complete degradation of the RNA target. The details of the conditions used in this assay are present in the Methods section in the original manuscript (lines 203-211), including composition of the lysis buffer and incubation times. Of note, we performed these experiments using naked RNA spiked into saliva only in Fig 1A as a proof of principle that RNA can bind the Whatman paper in the context of saliva. In Fig 1B we use synthetic (encapsulated RNA) viral particles; in subsequent Figures using saliva, samples were incubated with RNAlater to inhibit RNase activity. In Fig 4 we show how we can detect endogenous human mRNA (as positive control of the assay); and in Fig 5 and Fig 6 we show how our assay detects SARS-CoV-2 RNA in real COVID-19 patients. Kim, D., Lee, J.-Y., Yang, J.-S., Kim, J.W., Kim, V.N, and Chang, H. The Architecture of SARS-CoV-2 Transcriptome. Cell 181:914-921 (2020). Liu, W., and Tsou, C.L. Activity change during unfolding of bovine pancreatic ribonuclease A in guanidine. Biochimica et Biophysica Acta 916:455-464 (1987). 3) In describing the LoD the authors say that their assay is ">50X more sensitive than other recent saliva LAMP assays" which is a bit unfair considering this is contrived samples, data in Fig. 5A doesn't really indicate a "50X" sensitivity improvement. Plus there are a lot of LAMP assays, picking 1 for LOD comparison doesn't quite justify a claim of superiority to "other assays". We have revised this section according to this comment to not refer to a 50X improvement in sensitivity. Reviewer 2: The Paper would greatly benefit from adding statistics throughout the paper and the statistics should be described in detail in the material and methods. Thank you for the suggestion. We have now increased our sample sizes in many parts of the paper and added statistical tests throughout (in main text and in figures), all of which support our observations and previous conclusions. We have a new section in the Methods explaining the statistical tests and the sample sizes. We also revised the data in the automatic quantification of WHotLAMP results using image analysis to remove some technical replicates. This removal did not change the non-overlapping distributions of hues between positive and negative samples and this difference in distributions in highly significant (P<0.0001). It is not clear how many samples that has been analyzed, there is a mention of 20/20 positive samples and then 21/21. A table of the patient/sample characteristics detailing the number of positive samples, the severity of disease, negative controls and other diseases would greatly benefit the paper. We have included a new Supplementary Table with details on numbers of COVID-19 patients tested, the severity of disease and other clinical details. We have also clarified throughout the revised manuscript the number of samples used in each experiment and also in a new section on Statistical analysis in the Methods. Attachments Attachment Submitted filename: 20210830_response_to_reviewers.docx https://doi.org/10.1371/journal.pone.0257464.r002
2 Sep 2021 Decision Letter - Etsuro Ito, Editor WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva PONE-D-21-23691R1 Dear Dr. Bendesky, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Etsuro Ito Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0257464.r003
Formally Accepted
7 Sep 2021 Acceptance Letter - Etsuro Ito, Editor PONE-D-21-23691R1 WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva Dear Dr. Bendesky: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Etsuro Ito Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0257464.r004

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