PONE-D-20-34741

Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. 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: 1. More references should be added. For example, references related to the reagent choices used in this work should be added.

2. I would like to see more justifications in using different sample input and elution volume among the three methods. Adding more volume in the MM in house method, of course, can improve the total yield. An better approach is using standardized input and elution volume across the three methods.

3. The authors claimed lysis buffer was not used in the two in-house methods and there should be more discussions on why the traditional GTC lysis buffer is not needed but still was able to deliver good yield. However, it is probably has something to do with the samples were already mixed with GTC and heated before being used in the extraction process (Sample collection stage, lines 59-65). Therefore, it is incorrect to suggest that the in-house protocols lack efficient lysis steps.

4. Addition discussion on risk of cross-contamination on the OTC platform should be discussion. Did the authors use negative samples along with positive samples in the same run?

5. Overall, the results of the study is not unexpected. In-house reagents are expected to be cheaper than reagent kits bought from vendor. Also, method using large input can compensate extraction yield. The impact of this work is on the value of using a open-source, less expensive, device to perform sample preparation. I would like to see how much time was spent on programming the device before it was ready to collect data for this study. In addition, if the device is later used for master mix preparation, will false positive arise?

Reviewer #2: Lázaro-Perona et al. describe open-source, bead-based RNA extraction methods that are significantly less expensive than the standard MagMAX kit used for SARS-CoV-2 diagnostics. These methods perform comparably to the MagMAX kit when evaluated using a large collection of 141 SARS-CoV-2 positive clinical samples. The authors’ alternative RNA extraction methods could reduce unnecessary reliance on expensive robotic systems and proprietary RNA extraction reagents for SARS-CoV-2 testing, making this an extremely useful advance. I recommend that this work be published with minor revisions.

Major comments:

1. It would be helpful to provide a more detailed, step-by-step description of the different protocols used. Instead of saying that the manufacturer’s instructions were followed for the MagMAX kit, it would be worth listing the steps explicitly. The manufacturer’s protocol may change in the future, and so it is important to record the current protocol at the time that this work was performed. It was also a bit difficult to follow the description of the two in-house protocols. Perhaps it would be clearer to format each protocol as a numbered list of steps. The authors should provide the product information for the 96-well plates and 96-well magnet that they used. How long were the plates kept on the magnet to pull the beads to the side, and how long were the beads left to air dry after the second ethanol wash?

2. In the experiment described in the first Results section, the in-house methods showed lower fractional recovery of nucleic acid than the MagMAX protocol. Because these experiments were performed with short pieces of DNA rather than viral RNA, it is unclear whether these measurements are informative. If possible, it would be best to repeat these experiments using viral RNA (e.g., pooled leftover RNA from clinical samples).

3. Fig. 1 is a bit difficult to interpret at a glance, and it would help to have a summary panel showing the overall percentage of samples that tested positive using each protocol (i.e., 114/141 = 81%, 111/141 = 79%, and 118/141 = 84%).

Minor comments:

1. It should be stated in the methods what concentrations of guanidine isothiocyanate and carrier RNA were added to the samples.

2. Line 67: “two equipments” isn’t grammatically correct. Perhaps say “two pieces of equipment” or “two liquid handling robots”.

3. In the “Statistical analysis” section, they say that they used the Wilcoxon signed-rank test because the Ct data were not normally distributed, but then say in the next sentence that they used a paired t-test (which assumes normality). Please clarify.

4. Lines 191 and 203: I think they mean “investment” rather than “inversion”

5. They mention that the protocols “lack an efficient lysis step”, unlike the MagMAX kit. Did they include a proteinase K incubation step in the MagMAX protocol, and might adding this step to their protocol improve its performance?

Reviewer #3: The manuscript entitled “Evaluation of two automated low-cost RNA extraction protoocls for SARS-CoV-2 detection” submitted by Minorance et. al compares multiple extraction methods for COVID-19 testing including 2 automated lab developed protocols. In the manuscript the authors used a magnetic bead base extraction with the Opentrons OT-2 liquid robot and the MagMAX express system. Comparison of the assays was performed first by a dilution series of a positive control and later by testing of 141 SARS-CoV-2 positive patient samples. The QC testing demonstrated similar efficiencies between the in house protocols to the industry method. When testing of the 141 clinical samples 123 were positive by at least one extraction method and 18 were negative on all three methods. The authors suggested this is due to degradation of specimens. Finally, the authors compared overall cost and demonstrated cost effectiveness of the lab developed extractions due to reagent cost. Overall, the study was well written and adds to the data about methods for improving COVID-19 testing during the pandemic. Extractions are some of the most time consuming and limiting steps and use of more automation should help improve laboratory workflows. There are a few modifications needed:

Major Comments:

- The data for efficiency is very interesting, but it would be helpful to add a table demonstrating QC concentration followed by the number of tests that were detected with that extraction method *similar to LoD result.

- It is a bit concerning that there were so many FN results from the 141 samples tested. What were the storage conditions of the specimens, were they stored at 2-8C or frozen and how long. Was the standard of care data extracted using one of these extraction methods?

- What was the transport media used for the specimens and did this have any effect if it was multiple media types.

Minor Comments

- Ln 98-100 What was the concentration of the viral material used and to save needing to look it up, was this a capsulated RNA, inactivated virus, or free RNA?

- Add y-axis legend for Figures 1 and 2

Reviewer #4: Lázaro-Perona et al. have compared two automated RNA extraction protocols for SARS-CoV-2 qPCR detection. There are few aspects that should be clarified for the readers to enable them to repeat the protocol and utilize it.

There has been previous work on automated RNA extraction protocols for SARS-CoV-2 incl. those utilizing alternative reagents (doi:10.1261/rna.076232.120). It will be pertinent to briefly mention some or refer to reviews on same for readers to obtain a perspective of this work.

Please provide link to protocol for MagMAX CORE Nucleic Acid Purification kit (MMkit). Is the 500 uL sample volume compatible with protocol recommendations? For instance, is there anything in the MMkit protocol (volumes, steps, use of GTC or other finer details) that is different from protocol used in the paper.

Please give details of exact reagents used for replication. For instance, viral transport medium (specification/manufacturer etc. or constituents if lab made).

"MagMAX TM with the same script as the commercial kit and with the reagents used in the OT-2 method"- What is meant by same script? Same volumes, protocol. It might be good to describe this protocol & volumes at least in supporting info for clarity. For instance, it mentions "a final volume of 500 uL for the OT-2in-house and 1000 uL for the MMin-house"- Does different volumes mean different concentrations here? For equivalent comparison why was the volume increases for MMin-house esp. since MB volume used is same (40 uL)? Based on this your extraction efficiency will be different? Why is it 200 uL for MMkit if the protocol is same as MMin-house? The protocols have differences in use of Lysis/Binding

buffer & Isopropanol. Do these affect the efficiencies or explain why the comparison of efficiencies are valid if these details in protocols are different. Maybe explain in introduction that in-house protocols are "extraction free protocols" or direct sample addition protocols.

In line 100, "mixing 10 ul of the positive control with 490 ul of viral transport medium and 500 ul of GTC". So for the 3 different protocols do you take 200, 250 & 500 ul of sample and mix with corresponding volume ration of GTC? Or are these volumes only relevant for clinical samples? If the dilution is different is expected concentration of RNA different?

What is the starting conc. of the DNA positive control TaqMan 2019-nCoV Control Kit v1 ?

Line 103-"All mock samples were prepared in triplicate"-Is that n=3 for each platform or is it n=1 for each? i.e were 3 samples run on each platform or one. Please clarify this.

"1:100, 1:1000 and 1:10000 respectively."- Assuming this is volume percent?

"corrected by the dilution factor and the amount of initial sample used in each protocol (Ctpc) (R = 2-ΔCt=2-(Ctss-Ctpc))."- I am assuming this is key line which explains how different initial sample volumes are corrected for concentration? Maybe clarify it or elaborate the eqn. for better understanding.

Line 115-"positive control with the qPCR conditions recommended by the manufacturer." - Please cite the protocol if available online or if published.

Line 128- "Efficiencies of the extraction protocols"-Please provide data used to calculate the extraction efficiency in tabular or other form (maybe in supporting data if not relevant to main text).

Line 85-"the mixtures are incubated for 5 minutes at room temperature"-Is there any vortexing or mixing.

Line 118-What volumes were dispensed for each of the protocols?

Line 153-"probably due to sample degradation during the collection period"-Did the human RNaseP genes get detected in these -ve samples? What was the original test done to determine positive status of the collected 141 SARS-CoV-2 positive nasopharyngeal swabs?

"The 18 samples that showed discrepancies between methods had high Ct values"- What samples are being referred here? What is nature of discrepancies?

"method used in 43%, 49% and 49% of the samples"-Does it mean all of the 3 targets were detected? Please clarify.

"and was irrelevant for the diagnostic"-Please explain this statement

"Samples with multi-target amplification"-Please explain multi-target amplification (amplifying at least 2 or more of the genes?)

"probably because in-house protocols have a larger sample starting volume that"- Please explain this statement. Sample volumes will have different concentrations?

"the presence of mucus can preclude the RNA extraction or inhibit the PCR reaction"-Please cite a reference for this statement.

Line 27-"designed to be use with"- used with

Line 67-"we used two equipments"-noun equipment does not have a plural form

Line 77-"as ethanol absolute"- such as ethanol or correct the sentence

Line 190 "OT-2 protocol for 96 samples resulted 40€ more expensive than the MMin-house, the"-Please correct sentence

Line 191-"required initial inversion is significantly lower, being approximately 10.000€ for the"-Inversion might be wrong word. Is it 10,000€ & 50,000€.

"The protocol requires few hands-on time" & "requires a lower inversion"- Please correct sentence

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection

PONE-D-20-34741R1

Dear Dr. Mingorance,

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.

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A. M. Abd El-Aty

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. 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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The authors have answered each of my comments and the comments from the other authors. I have no additional suggestions for the author.

Reviewer #4: All the review comments and detailed protocol descriptions have been well incorporated. The change incorporated will enable readers to reproduce the results described in the protocol.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Harikrishnan Jayamohan