Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2021 Feb 16;16(2):e0246302. doi: 10.1371/journal.pone.0246302

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

Fernando Lázaro-Perona 1,#, Carlos Rodriguez-Antolín 1,#, Marina Alguacil-Guillén 1, Almudena Gutiérrez-Arroyo 1, Jesús Mingorance 1,*, Julio García-Rodriguez 1; on behalf of the SARS-CoV-2 Working Group1,
Editor: A M Abd El-Aty2
PMCID: PMC7886139  PMID: 33591986

Abstract

Background

Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated.

Methods

One hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested in parallel with a commercial kit that uses the MagMAXTM system (MMkit). Nucleic acid extraction efficiencies were calculated from a SARS-CoV-2 DNA positive control.

Results

No significant differences were found between both in-house protocols and the commercial kit in their performance to detect positive samples. The MMkit was the most efficient although the MMin-house presented, in average, lower Cts than the other two. In-house protocols allowed to save between 350€ and 400€ for every 96 extracted samples compared to the commercial kit.

Conclusion

The protocols described harness the use of easily available reagents and an open-source liquid handling system and are suitable for SARS-CoV-2 detection in high throughput facilities.

Introduction

The SARS-CoV-2 pandemic has called for the use of massive qPCR tests in order to detect positive cases and to trace contacts to stop community transmission. Prior to qPCR testing, clinical samples are usually subject to RNA extraction [1–4]. Given the number of samples tested every day, manual RNA extraction methods are unfeasible for most facilities and thus, automatic systems are widely used for this task [5–7]. As a drawback, automatic systems significantly increase the final costs, which can hinder massive testing in some areas. Moreover, due to the increased demand, stock shortage of extraction reagents has caused major delays in diagnostics.

In this work, two low-cost alternatives for automatic RNA extraction are described. The first one using the OT-2 system (Opentrons, New York, NY, USA), an open-source liquid handler robot capable of automating self-designed protocols, and the second using the rapid and easy-to-use nucleic acid extractor MagMAXTM Express-96 system (Thermo Fisher Scientific, Waltham, MA, USA). The later extracts up to 96 samples in 30 min, though it requires a previous manual dispensation of the reagents, magnetic beads and samples in 96-well plates adding 30 min. As an alternative, the OT-2in-house protocol can process up to 48 samples in 104 min in a fully automated manner.

Methods

Sample collection

During ten days 141 consecutive SARS-CoV-2 positive nasopharyngeal swabs with viral transport medium (Deltalab, Barcelona, Spain) that had been stored at 4°C were collected. Before processing, the samples were inactivated by mixing 500 μL of the viral medium and 500 μL of 4M guanidine Isothiocyanate (GTC) (Qiagen, Hilden, Germany) with 5 μg/mL carrier RNA, followed by heating the samples 2 min at 80°C and a short vortex mixing.

Equipment and reagents

Automatic extraction of nucleic acids was done using two systems: the MagMAXTM Express-96 Deep Well Magnetic Particle Processor (King Fisher Instrument, Thermo Fisher Scientific, Waltham, MA, USA) and the open system OT-2 (Opentrons, New York, NY, USA) with a GEN1 magnetic module (Opentrons, New York, NY, USA) and an in-house protocol. MagMAX™ Express 96 plates and Deep Well plates (Thermo Fisher Scientific, Waltham, MA, USA) were used with the two systems.

Nucleic acid extraction was done with three methods: 1) Using the MagMAX TM with the commercial MagMAX CORE Nucleic Acid Purification kit (MMkit) (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer instructions; 2) The OT-2 system with generic reagents (OT-2in-house) such as ethanol (Emsure®, Merck KGaA, Darmstadt, Germany), 2-Propanol (Emsure®, Merck KGaA, Darmstadt, Germany), Elution Buffer (Omega BIO-TEK, Norcross, GA, USA), Nuclease free water (AmbionTM, Thermo Fisher Scientific, Waltham, MA, USA) and magnetic beads (Mag-Bind® TotalPure NGS, Omega Bio-Tek, Norcross, GA, USA); 3) MagMAX TM with the same protocol as the commercial kit but the reagents used in the OT-2 method (MMin-house). In-house protocols are a modification of the procedure described by Hui He et al. [8]. Briefly, inactivated respiratory samples are mixed in a 1:1 proportion with isopropanol to a final volume of 500 μl for the OT-2in-house and 1000 μL for the MMin-house, 40 μL of magnetic beads are added and the mixtures are incubated for 5 min at room temperature. Next, magnetic beads are pulled to one side of the tubes with a magnet and supernatant is discarded. Magnetic beads are then washed two times with 500 μL of freshly prepared ethanol 70%. After the second wash, the ethanol 70% is discarded and the magnetic beads are air dried at room temperature. Finally, the beads are resuspended in 100 μL of the elution buffer and separated with the magnet again to recover the eluted viral RNA (Table 1).

Table 1. Steps, reagents and volumes (μL) used in the three protocols evaluated.

Step1 (Reagent mix) Step2 (Wash) Step3 (Wash) Step4 (Elution)
Sample Lysis/Binding buffer Isopropanol absolute Magnetic Beads Wash 1 buffer* Wash 2 buffer* Elution buffer
MMkit 200 700 - 30 500 500 90
OT-2in-house 250 - 250 40 500 500 100
MMin-house 500 - 500 40 500 500 90
Open in a new tab

*The MMin-house and OT-2in-house protocols use ethanol 70% in the two washing steps.

Protocol design and validation

MMkit protocol:

(Taken from https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0015944_MagMAXCORE_NA_Kit_UG.pdf)

  1. Prepare two Deep well plates, one with 500μL of MagMAX™ CORE Wash Solution 1 per well, and another one with 500μL of MagMAX™ CORE Wash Solution 2 per well. Prepare a MagMAX™ Express 96 microtiter plate with 90μL of MagMAX™ CORE Elution Buffer per well.

  2. Prepare a mixture of 20μL of MagMAX™ CORE Magnetic Beads and 10μL of MagMAX™ CORE Proteinase K per sample (beads/PK mix).

  3. Prepare a mixture of 300μL MagMAX™ CORE Lysis Solution and 300μL MagMAX™ CORE Binding Solution per sample (Lysis/Binding solution).

  4. Dispense in a Deep well plate 30μL of beads/PK mix, 700μL of Lysis/Binding solution and 200μL of sample per well.

  5. Put all the plates in the system and run the script MagMAX_CORE_KF-96.

MMin-house protocol:

  1. Prepare two Deep well plates with 500μL of Ethanol 70% per well. Prepare a MagMAX™ Express 96 microtiter plate with 90μL of Elution Buffer (Omega BIO-TEK, Norcross, GA, USA) per well.

  2. Dispense in a Deep well plate 40μL of magnetic beads (Mag-Bind® TotalPure NGS, Omega Bio-Tek, Norcross, GA, USA), 500μL of Isopropanol and 500μL of sample per well.

  3. Put all the plates in the system and run the script MagMAX_CORE_KF-96.

OT-2in-houseprotocol:

  1. Dispense in a Deep well plate 40μL of magnetic beads (Mag-Bind® TotalPure NGS, Omega Bio-Tek, Norcross, GA, USA), 250μL of Isopropanol and 250μL of sample per well. Mix by pipetting five times and incubate 5 min at room temperature.

  2. Activate the GEN1 magnetic module 4 min.

  3. Collect the supernatant and discard.

  4. Add 500μL of Ethanol 70%, collect and discard.

  5. Add 500μL of Ethanol 70%, collect and discard.

  6. Air dry for 4 min.

  7. Turn off the GEN1 magnetic module and add 100μL of Elution Buffer (Omega BIO-TEK, Norcross, GA, USA).

  8. After 30 sec turn on the GEN1 magnetic module.

  9. After 90 sec collect the supernatant and transfer to a 96-well microtiter plate.

The sample input was the maximum volume allowed by the automatic pipetting systems and the volumes of the other reagents and therefore was different for the three methods

OT-2in-house protocol was written in Python according to the Opentrons instructions. The scripts have been deposited in the GitHub repository (https://github.com/HULPopentrons/RNA_extraction_OT2opentrons).

To validate the performance of the in-house protocols for nucleic acid extraction, simulated samples were made with standard, low and very low viral loads using the DNA positive control TaqMan 2019-nCoV Control Kit v1 (Thermo Fisher Scientific, Waltham, MA, USA). The Control Kit comes at a concentration of 1 x 104 copies/μL. The standard viral load sample was prepared by mixing 10 μL of the positive control with 490 μL of viral transport medium and 500 μL of GTC. The low and very-low viral loads were prepared in the same manner but using ten-fold serial dilutions of the positive control. All mock samples were prepared in triplicate and processed in parallel with the OT-2in-house, the MMkit and the MMin-house and then tested by qPCR with the TaqMan 2019-nCoV Assay Kit v1. Final concentration of the positive control in the simulated samples with the standard, low and very-low viral loads was 1 x 106 copies/mL, 1 x 105 copies/mL and 1 x 104 copies/mL respectively. Negative controls were included in all the runs.

Nucleic acid extraction efficiency was calculated by comparing the Ct values of the positive controls in the simulated samples (Ctss) with the Ct values of the positive controls prepared directly from the stock correcting the amounts to match the dilution factor and the amount of initial sample used in each protocol (Ctpc) (R = 2-ΔCt = 2-(Ctss-Ctpc)).

qPCR

Nucleic acid amplification of the SARS-CoV-2 viral RNA was done using the TaqMan 2019-nCoV Assay Kit v1 that targets the orf1ab, spike (S), nucleocapsid (N) and human RNaseP genes and the TaqMan 2019-nCoV Control Kit v1 as positive control with the qPCR conditions recommended by the manufacturer (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0019096_TaqMan2019nCoVAssayKit_PI.pdf). All qPCR assays were performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). To reduce inter-assay variability, extracted nucleic acid samples were dispensed automatically in qPCR strips with another OT-2 module.

Statistical analyses

The three protocols were compared pairwise. McNemar’s test was used to compare their performance in assigning samples as positive or negative. Ct values are not distributed normally, therefore the Wilcoxon Signed-Rank test was used for comparing the Ct values of each target. For each target only the samples that were amplified by the three methods were considered. Median confidence intervals were calculated with a bootstrap method.

All the statistical tests were performed with the IBM SPSS Statistics 24.0.0.0 package (SPSS Inc., Chicago, IL, USA).

Results

Efficiencies of the extraction protocols

Nucleic acid extraction efficiencies of the two in-house protocols were compared with the MMkit protocol by extracting the mock samples prepared with the SARS-CoV-2 positive control that simulate different viral loads. The MMkit and OT-2in-house protocol successfully amplified the orf1ab, S and N targets in all mock samples with the standard viral load. For these samples, the mean extraction efficiency of all replicates and genes was 33.9% (SD: 13.8) for the MMkit, 19.6% (SD: 2.2) for the OT-2in-house protocol and 16.0% (SD: 5.03) for the MMin-house.

In the low viral load mock samples, the MMkit failed to amplify the S target in all samples and the N target in one of the replicates while the OT-2in-house and MMin-house detected all the genes. Finally, the MMkit and OT-2in-house protocols failed to amplify the very-low viral load samples, except for one replicate in which the N gene could be amplified with the OT-2in-house protocol but the MMin-house could detect the positive control in all samples, one of them with the three targets, one with them with the orf1ab and N targets and the last one with the orf1ab target (S1 Table).

Nucleic acid extraction from clinical respiratory samples

The 141 positive clinical samples collected were subjected to nucleic acid extraction in parallel with the MMkit, the OT-2in-house, and the MMin-house methods and the eluted SARS-CoV-2 RNA was tested by qPCR. Positive/negative results and amplification cycle threshold values (Ct) for each target were registered. According to the manufacturer’s instructions samples were considered positive when at least one of the three-targeted regions amplified with a Ct<40. Negative controls included in all the runs yielded in all cases negative PCR results.

Of the 141 samples, 123 were positive for SARS-CoV-2 by at least one extraction method while 18 were negative by all three methods. This was probably due to sample degradation during the collection period, because they had been stored for about 48 h at 4°C [9]. By method, 114 samples tested positive using the MMkit, 111 with the OT-2in-house and 118 with the MMin-house. Pairwise comparisons found no significant differences in their performances for detecting SARS-CoV-2 (MMkit Vs OT-2in-house, P = 0.5465; MMkit Vs MMin-house, P = 0.3865; OT-2in-house Vs MMin-house, P = 0.0961). Eighteen samples were negative by some of the three methods. These had high Ct values, with median values of the orf1ab and N targets of 38.68 and 38.19 respectively (in those samples the S target was not amplified by any method). Pairwise linear regression and correlation analyses comparing all methods and targets showed R2 values between 0.85 and 0.95 and Bland-Altman analyses showed the agreement to be uniform through the whole Ct range (S1 Fig).

The three targets (orf1ab, N and S) were detected in 43% of the samples extracted with MMkit, 49% of those extracted with OT-2in-house and 49% with MMin-house (Fig 1). In 34%, 39% and 30% the orf1ab and N targets were detected, and in 19%, 10% and 17% only the N target was amplified. Few samples were considered positive by the amplification of either the orf1ab target alone (6), the orf1ab + S (1), or the N + S (5) targets, and no sample had the S gene as the only positive marker. In fact, the S target in this assay had an evident lack of sensitivity and would be irrelevant to take diagnostic decisions. Samples with two or three targets detected were 87% (97 samples) with the OT-2in-house protocol, 82% with the MMin-house (95 samples) and 79% with the MMkit (90 samples).

Fig 1. Percentages of SARS-CoV-2 samples positive for the orf1ab, S and N targets with the MMkit, OT-2in-house and MMin-house methods.

Fig 1

Open in a new tab

The numbers over the bars indicate the percentage of positive samples for each target. Overall, 114 (81%) samples were positive with MMkit, 111 (79%) with OT-2in-house and 118 (84%) with MMin-house.

When considering paired samples, no significant differences were found in the Cts between the MMkit and the OT-2in-house protocol in the Ct values for the orf1ab (P = 0.437), N (P = 0.686) and S (P = 0.794) targets (Fig 2). The MMin-house protocol did present significantly lower Cts in all targets compared to the MMkit (orf1ab; P< 0.00001, N; P< 0.00001, S; P = 0.00148) and the OT-2in-house (orf1ab; P< 0.00001, N; P = 0.00008, S; P = 0.00252).

Fig 2. Boxplot showing the distribution of the Ct values obtained for the orf1ab, S and N targets with the MMkit, OT-2in-house and MMin-house methods.

Fig 2

Open in a new tab

The y-axis shows the amplification cycle (Ct). The number of data points is shown over each data set.

The median amplification cycle (CI:95%) of the orf1ab target using the MMkit, the OT-2in-house and the MMin-house were, respectively, 35.53 (33.82–36.36), 35.58 (34.33–36.21) and 34.8 (33.71–35.29). For the S gene 30.16 (28.56–33.08), 31.32 (29.22–32.88) and 31.07(29.36–32.90) and the N target 34.83 (33.93–35.97), 34.64 (33.42–35.29) and 34.28 (33.52–35.10).

Extraction costs and hands-on time

Nucleic acid extraction of 96 samples using the OT-2in-house protocol had an overall cost of 107€ (37€ reagents and 70€ labware) with a hands-on time of 10 min and an extraction time of 3hours and a half (the script handles 48 samples in each run so it has to be completed two times). The MMin-house cost was 66€ (37€ reagents and 29€ labware) and the MMkit, 472€ (443€ reagents and 29€ labware). Both protocols had a hands-on time of 40 min and an extraction time of 30 min. Notably, although the OT-2 protocol for 96 samples was 40€ more expensive than the MMin-house, the required initial investment is significantly lower, being approximately 10,000€ for the OT-2 system (including modules and pipettes) and 50,000€ for the MagMAXTM system.

Discussion

In this work, two methods are presented for low-cost viral RNA extraction and detection of SARS-CoV-2 in clinical samples with a performance comparable to that of a commercial kit. Although in-house protocols extracted the DNA control less efficiently than the commercial kit, in clinical samples the overall sensitivity was not affected, probably because in-house protocols use larger sample starting volumes that compensate the lower efficiency.

Setting up the OT-2 system for RNA extraction required intensive programming for a non-experienced team but provided a cost-effective alternative capable of extracting 48 samples in a single run. The scripts used in this work were uploaded to the open access software repository GitHub, where many protocols for these and similar applications can be found. This should help other workers to avoid or reduce the programming steps. The protocol demands little hands-on time and uses easily available reagents. In addition, the equipment requires a lower investment compared to other extraction systems, making it suitable for middle to low resource facilities. The MagMAXTM Express-96 is a fast, semi-automatic equipment capable of extracting 96 samples per run. The MMkit provides reagents for inactivation, lysis, washing and elution and can be used for different matrices and samples types at a competitive cost. On the other hand, the MMin-house protocol takes advantage of the semi-open approach of this system by substituting the commercial solutions by other chemicals making it more cost effective. In both cases, preparing the deepwell plates with buffers and samples must be done manually, increasing the total extraction time and the manipulation error risk. The main drawback of the two in-house protocols comes when handling viscous samples, the presence of mucus, highly viscous polysaccharides, leukocytes, erythrocytes, hemoglobin, proteases and cell detritus with high amounts of cellular nucleic acids can preclude the RNA extraction or inhibit the PCR reaction [10–12]. To partially overcome this problem, samples can be heated and vortexed or subjected to a centrifugation prior to extraction, though this would increase the hands-on and response times. When using the MMin-house, the eluted samples may have in some cases traces of the magnetic beads though this did not affect the performance of the RT-PCR reactions.

With the three systems negative controls were in all cases negative, indicating that cross-contamination is not an issue when working with primary samples in these systems. Nevertheless, same as with non-automated systems, precautions should be taken to keep pre-PCR and post-PCR operations separated. If an OT-2 module is used to set-up post-PCR reactions (eg. sequencing) it should not be used to extract nucleic acids from samples unless it is thoroughly decontaminated.

Study strength and limitations

The major strength of the work is that the methods were tested with real samples in a clinical microbiology laboratory. The comparison between methods was not systematic, and this is an important limitation. In fact, the design was intended to optimize the results for each system, and so the inputs were different. A systematic exploration of the input sample volumes, as well as other reagent volumes would be helpful and informative, but it was beyond the aim of this work that was to compare the performance of the systems in a clinical laboratory environment with real samples.

Conclusion

In summary, the two in-house nucleic acid extraction methods presented here are efficient for the high throughput diagnosis of SARS-CoV-2 at fraction of the costs of other commercial kits without losing sensitivity.

Supporting information

S1 Table. Cts obtained with the simulated samples at three different concentrations (Ctss) and Cts of the diluted controls used to calculate the efficiency (Ctpc).

(n.d. not detected).

(TIF)

Click here for additional data file. (750.7KB, tif)
S1 Fig. Bland-Altman plot showing the agreement between the three methods with three marker genes.

The same set of samples was extracted with the three methods and analyzed with a commercial PCR targeting three marker genes. The horizontal axes show the average Cts, and the vertical axes show the difference between Cts for each sample with the two methods indicated. The horizontal continuous line marks the average difference, and the discontinuous lines indicate the 95% limits of agreement (average difference ± 1.96 standard deviation of the difference).

(TIF)

Click here for additional data file. (601.9KB, tif)
S1 Data

(XLSX)

Click here for additional data file. (23.4KB, xlsx)

Acknowledgments

We are grateful to the COVID Warriors organization (www.covidwarriors.org) for donating the Opentrons OT-2 stations.

The authors also acknowledge the additional members of the SARS-CoV-2 Working Group: María Dolores Montero-Vega, María Pilar Romero, Silvia García-Bujalance, Emilio Cendejas Bueno, Carlos Toro-Rueda, Guillermo Ruiz-Carrascoso, Fernando Lázaro Perona, Iker Falces-Romero, Almudena Gutierrez-Arroyo, Patricia Girón de Velasco-Sada, Mario Ruiz-Bastián, Marina Alguacil-Guillén, Patricia González-Donapetry, Gladys Virginia Guedez-López, Paloma García-Clemente, María Gracia Liras Hernandez, Consuelo García-Sanchez, Miguel Sánchez-Castellano and Sol San José-Villar. Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ, Paseo de La Castellana 261, 28046 Madrid, Spain.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

  • 1.Merindol N, Pépin G, Marchand C, Rheault M, Peterson C, Poirier A, et al. SARS-CoV-2 detection by direct rRT-PCR without RNA extraction. J Clin Virol. 2020;128: 104423 10.1016/j.jcv.2020.104423 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Esbin MN, Whitney ON, Chong S, Maurer A, Darzacq X, Tjian R. Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection. RNA. 2020;26: 771–783. 10.1261/rna.076232.120 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bain W, Lee JS, Watson AM, Stitt‐Fischer MS. Practical Guidelines for Collection, Manipulation and Inactivation of SARS‐CoV‐2 and COVID‐19 Clinical Specimens. Curr Protoc Cytom. 2020;93 10.1002/cpcy.77 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Wang Y, Song W, Zhao Z, Chen P, Liu J, Li C. The impacts of viral inactivating methods on quantitative RT-PCR for COVID-19. Virus Res. 2020;285: 197988 10.1016/j.virusres.2020.197988 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Ambrosi C, Prezioso C, Checconi P, Scribano D, Sarshar M, Capannari M, et al. SARS-CoV-2: Comparative analysis of different RNA extraction methods. J Virol Methods. 2021;287: 114008 10.1016/j.jviromet.2020.114008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Klein S, Müller TG, Khalid D, Sonntag-Buck V, Heuser AM, Glass B, et al. SARS-CoV-2 RNA extraction using magnetic beads for rapid large-scale testing by RT-qPCR and RT-LAMP. Viruses. 2020;12 10.3390/v12080863 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ransom EM, Potter RF, Wallace MA, Mitchell KF, Yarbrough ML, Burnham CAD, et al. Comparison of extraction methods and thermocyclers for SARS-CoV-2 molecular detection using clinical specimens. J Clin Microbiol. 2020;58: 1–3. 10.1128/JCM.01622-20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.He H, Li R, Chen Y, Pan P, Tong W, Dong X, et al. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections. Sci Rep. 2017;7: 45199 10.1038/srep45199 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Li L, Li X, Guo Z, Wang Z, Zhang K, Li C, et al. Influence of Storage Conditions on SARS-CoV-2 Nucleic Acid Detection in Throat Swabs. J Infect Dis. 2020;222: 203–205. 10.1093/infdis/jiaa272 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol. 1997;63: 3741–3751. 10.1128/AEM.63.10.3741-3751.1997 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Al-Soud WA, Jönsson LJ, Rådström P. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol. 2000;38: 345–350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Jaulhac B, Reyrolle M, Sodahlon YK, Jarraud S, Kubina M, Monteil H, et al. Comparison of sample preparation methods for detection of Legionella pneumophila in culture-positive bronchoalveolar lavage fluids by PCR. J Clin Microbiol. 1998;36: 2120–2122. 10.1128/JCM.36.7.2120-2122.1998 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

A M Abd El-Aty

1 Dec 2020

PONE-D-20-34741

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

PLOS ONE

Dear Dr. Mingorance,

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.

As appended below, the reviewers have raised major concern/critique and suggested further justification/work to consolidate the findings. Do go through the comments and amend the MS accordingly. Furthermore

1- Avoid using "We, our". Use impersonal phrasing throughout the text

2- Full vendor details should include company, city (state), and country. Please amend and be consistent throughout the MS

3- Please use uL, mL, L throughout the text

4- The P letter for statistical value should be uppercase-italic face letter throughout the text

5- Please use min instead of minute throughout the text

6- What are the "Study strength and limitations". Please add this in a separate section headed as stated. It should be before conclusion section

7- Conclusion should be in a separate section headed as stated. What are the clinical relevance and future perspective. Please address them in this section as well

8- Abstract should be "structured one". i.e. it should include background, methods, Results and Conclusion

Please submit your revised manuscript by Jan 15 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.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

A. M. Abd El-Aty

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for including your ethics statement:  "The work was done with anonymous discarded remnants of clinical samples set to be destroyed. No previous data on the patients or the samples themselves was collected. The results of the assays performed had no impact in any way on patient management.".   

Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

4. One of the noted authors is a group or consortium [SARS-CoV-2 Working Group]. In addition to naming the author group, please list the individual authors and affiliations within this group in the acknowledgments section of your manuscript. Please also indicate clearly a lead author for this group along with a contact

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

**********

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

**********

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

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Feb 16;16(2):e0246302. doi: 10.1371/journal.pone.0246302.r002

Author response to Decision Letter 0

30 Dec 2020

Reviewer #1:

More references should be added. For example, references related to the reagent choices used in this work should be added.

- Done as requested. References 5-8 added.

I would like to see more justifications in strengths and limitations 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.

- The reviewer is right and there is room for further optimization of the in house methods, but the aim of the work was not to do a systematic comparison of the systems, but to evaluate the utility of the in house systems for the clinical microbiology laboratory in an emergency situation in which there was shortage of commercial kits, so we used the maximal sample volume allowed by each system. A paragraph along this line has been included in the section " strengths and limitations".

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.

- The reviewer is right. The text has been corrected.

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?

- Negative controls were included in all the runs. This information has been added to the text, as well as a paragraph on cross-contamination in the discussion.

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?

- Protocol programming took several days for a non-experienced team. The scripts were uploaded to the GitHub repository that contains an increasing number of open-access scripts to help workers to reduce or avoid programming. Once the programming is done, setting up the OT-2 module, even if some readjustments were needed, might take one or two days. This has been commented in the text.

There were no cross-contaminations when working with primary samples, nevertheless all the usual precautions taken when working with diagnostic PCR should be taken, so we routinely perform master mixes in a different module. Post-PCR operations (eg. NGS library preparation) should never be done in a module intended for pre-PCR operations. This has been commented in the text.

Reviewer #2:

Major comments:

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.

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?

- The information requested has been added to the text.

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).

- The reviewer is right, but as far as we know (communication from the company) the control DNA is a plasmid containing a synthetic construction with all the target sequences. They are not short pieces. The suggestion of the reviewer of doing the comparison with viral RNA is interesting and was discussed by us, but after seeing the results obtained with the samples we felt that it was not necessary. As commented to reviewer#1 the aim of the work was not to do a systematic comparison of the systems, but to evaluate the utility of the in house systems for the clinical microbiology laboratory in an emergency situation in which there was limited availability of commercial kits.

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%).

- This information has been added to the figure legend in order to make it more clear.

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

- Done as requested.

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

- Corrected

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.

- This was an error. Reference to the paired t-test has been deleted.

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

- Corrected

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?

- As pointed by reviewer #1 the statement was not correct. The text has been corrected. Proteinase K was included only in the MMkit method.

Reviewer #3:

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.

- A supplementary table has been added.

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?

- The standard of care was extracted with the MMkit and the PCR was the same, but the samples were processed immediately after inactivation with guanidinium chloride. The remaining inactivated material was stored at 4ºC for 24-48 hours until all the samples for this work had been collected. During this storage at 4ºC viral RNA may be lost in up to 30% of the samples. We have observed this phenomenon several times and have added a reference from another group documenting similar observations (ref. 9).

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

- Transport medium and swab were part of a kit commercialized by Deltalab. The reference to Deltalab that was after "swab" has been moved to the end of the sentence to make this more clear. The company does not disclose the composition of the medium, just that it contains bacterial and fungal growth inhibitors. During the collection period a single transport media was used.

- 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?

- As stated in the text the control DNA was plasmid. The clinical samples were real samples taken from patients and containing intact virus.

- Add y-axis legend for Figures 1 and 2

- Corrected

Reviewer #4:

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.

- Several references have been added.

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.

- The protocol has been described and a reference to the original pdf has been included. The MMkit is the same as the one described by the company.

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

- As stated above, viral transport medium and swab were part of a kit commercialized by Deltalab. The reference to Deltalab that was after "swab" has been moved to the end of the sentence to make this more clear. The company does not disclose the composition of the medium, just that it contains bacterial and fungal growth inhibitors. During the collection period this was the only transport media was used.

"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.

- The protocols have been included in the text to make it more clear. The same script means the same instrument program.

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?

- The protocols have been included in the text to make it more clear. Different volume means different starting volume. The samples were the same, so the starting concentrations were the same. This is discussed now in the strengths and limitations section.

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?

- Increasing the sample volume increases the amount of material to be extracted and therefore the final yield. MBs are far from saturation and have capacity to bind higher amounts of RNA, we do not think that extraction efficiency is different, but the final yield is higher.

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.

- The reviewer is right, the protocols are different and start with different amounts of sample. The aim of the work was not as much to do a systematic comparison of the systems as to evaluate the utility of the in house systems for the clinical microbiology laboratory, so we used the maximal sample volume possible in each system. A paragraph along this line has been included in the section " strengths and limitations".

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.

- The protocols were different, as stated above. Comparison of the efficiencies was intended to be used as a guideline.

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?

- The 200, 250 & 500 ul already include GTC.

If the dilution is different is expected concentration of RNA different?

- Dilution is the same, 1:1. As explained above, the starting amount is different.

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

- 1 x 104 copies/µL. This information has been included in the text.

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.

- It was one triplicate per dilution per platform.

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

- This is dilution. The text has been rewritten to clarify.

"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.

- The explanation was not clear. The positive controls were diluted to match the amounts introduced in the extraction in each protocol, so no further corrections are needed. The sentence has been rewritten to make it more clear.

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

- Done as suggested.

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).

- A supplementary table has been added.

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

- The machines mix by repeated pippeting. The protocols have been included in the manuscript and this is explicitly stated.

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

- To make it more clear the protocols have been included in the manuscript and the amounts are explicitly stated.

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 standard of care was extracted with the MMkit and the PCR was the same, but the samples were processed immediately after inactivation with guanidinium chloride. The inactivated material left after taking the sample for the standard of care was stored at 4ºC for 24-48 hours until the 141 samples were collected and used. During this storage at 4ºC viral RNA may be lost in up to 30% of the samples while the RNAseP is still positive. We have observed this phenomenon several times and have added a reference from another group documenting similar observations (ref. 9).

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

- 18 samples were negative by one of the three methods. The sentence has been rewritten to make it more clear.

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

- The sentence has been rewritten to make it more clear.

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

- The sentence has been rewritten to make it more clear.

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

- The sentence has been rewritten to make it more clear.

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

- The volumes are different and the concentrations are the same, therefore the starting amount of RNA would be different.

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

- Three references have been added (10-12).

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

- Done

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

- Done

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

- Done

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

- Done

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€.

- Done

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

- Done

Decision Letter 1

A M Abd El-Aty

18 Jan 2021

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.

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,

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

**********

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

**********

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

**********

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

**********

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.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Harikrishnan Jayamohan

Acceptance letter

A M Abd El-Aty

3 Feb 2021

PONE-D-20-34741R1

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

Dear Dr. Mingorance:

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. A. M. Abd El-Aty

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Cts obtained with the simulated samples at three different concentrations (Ctss) and Cts of the diluted controls used to calculate the efficiency (Ctpc).

    (n.d. not detected).

    (TIF)

    Click here for additional data file. (750.7KB, tif)
    S1 Fig. Bland-Altman plot showing the agreement between the three methods with three marker genes.

    The same set of samples was extracted with the three methods and analyzed with a commercial PCR targeting three marker genes. The horizontal axes show the average Cts, and the vertical axes show the difference between Cts for each sample with the two methods indicated. The horizontal continuous line marks the average difference, and the discontinuous lines indicate the 95% limits of agreement (average difference ± 1.96 standard deviation of the difference).

    (TIF)

    Click here for additional data file. (601.9KB, tif)
    S1 Data

    (XLSX)

    Click here for additional data file. (23.4KB, xlsx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES