Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes

ABSTRACT The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high-throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false-negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multitarget redundancy and an internal amplification control. A convenient and cost-effective target-specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-CoV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high-frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multitarget redundancy. When challenged with extracted human samples the multiplex assay showed 87% or better sensitivity (of 30 positive samples), with 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction (of 21 positive samples), and 100% specificity (of 60 negative samples). These results are further evidence that conventional laboratory methodologies can be leveraged at the point of care for robust performance and diagnostic stability over time. IMPORTANCE The COVID-19 pandemic has had tremendous impact, and the ability to perform molecular diagnostics in resource limited settings has emerged as a key resource for mitigating spread of the disease. One challenge in COVID-19 diagnosis, as well as other viruses, is ongoing mutation that can allow viruses to evade detection by diagnostic tests. We developed a test that detects multiple parts of the virus genome in a single test to reduce the chance of missing a virus due to mutation, and it is designed to be simpler and faster than typical laboratory tests while maintaining high sensitivity. This capability is enabled by a novel fluorescent probe technology that works with a simple constant temperature reaction condition.

, more replicates and dilutions are needed (using 3 genes mixed or intact viral RNA) in order to derive LOD at 95%, at least 10 replicates (best 22) with dilutions of RNA,200,100,50,20,10,5,2.5,, this can be done in one plate. Actually authors did not mention if this LAMP can be done in high-throughput plate fashion. Fourth, LAMP has cross-contamination issue to address, not clear how experiments were done in Fig 3b. Authors should test samples on same plate with some high viral titer and negatives (processed at same time from extraction, ideally blinded), rather than separately tested. More positive samples are needed for robust validation of performance (current 21). Lastly, IAC (as negative control) is useful, however the system lacks a sample matrix control like RP, so no way to know if NP sample is low quality; this potentially leads to false negatives since RT-LAMP may report as negative due to sub-optimal sample (but LAMP has no quality control for this). Is there any RP-failed samples (by PCR) for testing? Minor comments: 1. I wonder if some mutations in N gene such as position 28881 may affect this assay performance 2. The sensitivity should be reported as 90% in Abstract, not 100%; specificity of 100% is a bit suspicious, as UDP may have non-specificity. Maybe authors did all negatives together (if in this case), they should validate cross-contamination in plate format, mimicking clinical testing (in blind, high titer could be processed with low titer or negative samples together). 3. Line 53. "..diversity has rendered some NAATs susceptible to false negative results,", can authors explain to readers why genetic diversity lead to false negatives, maybe due to mutations in the target region of an assay, is it more susceptible for single gene target than multiple target design; give some examples of commercial assay affected by mutations (citations), e.g. S-drop out by B.1.1.7 using TaqPath. 4. Line 117. What is synthetic dsDNA gBlocks, N gene only, specify. 5. Line 126. Give details of OpenArray, is it Taqman Realtime PCR, which commercial qPCR kit was used or in-house developed. Also add columns of Ct value of N1 and N2 in Table S1. 6. Fig 3A is confusing, above 35 minutes are considered "Not detected"?
Reviewer #2 (Comments for the Author): In this paper, Kline et al. established a multiplex target-redundant RT-LAMP assay for SARS-CoV-2 detection using universal displacement probes, which enables the detection of various variants. My comments and concerns are: 1. Whether the on-going generation of various variants of SARS-CoV-2 affects diagnostic tests by RT-qPCR and/or RT-LAMP assays should be evaluated (or discussed). Are there any data or literatures to support this view? 2. Multiplex RT-LAMP system based on displacement FIP/BIP probes have been developed (BioTechniques 2012, 53:81-89;Anal. Chem. 2016, 88, 3562−3568). In this study, the authors using universal displacement probe linking/binding to loop primer. In fact, this strategy (based on loop primers) was not novel (had been published previously (Anal. Chem. 2018, 90, 4741−4748;PLoS ONE 2021, 16(3): e0248042: this paper focused on SARS-CoV-2 detection). The authors did not mention the previous papers. On the other hand, the principle is similar between the two strategies (based on FIP/BIP and FL/BL primers). The authors should mention and discuss (compare) the advantages and disadvantages of both methods. 3. Mismatch/variant-tolerant LAMP method had been developed previously for efficient detection of highly variable RNA viruses (e.g. HIV-1) (Front Microbiol 2019，10:1056Analyst 2021, 146: 5347-5356), and showed well performance. The authors should mention and discuss (compare) the advantages and disadvantages of two kinds of strategies for detection of highly variable viruses/targets. This method was also used for detection of SARS-CoV-2 (Virologica Sinica，2020, 35:344-347). 4. The performance of this multiplex RT-LAMP assay on various mismatches between primers and templates should be evaluated like previous studies (Front Microbiol 2019，10:1056Analyst 2021, 146: 5347-5356). 5. In the clinical evaluation experiments, 30 SARS-CoV-2-postive samples were used. First, the ample size was too small. Second, did these samples contain some SARS-CoV-2 variants? Third, clinical samples used in the evaluation should contain at least two-three most common variant. If there is some difficulty in the collection of clinical samples carrying variants, simulated samples with in vitro synthesized/transcribed variants' RNA should be used. 6. In addition, the gold standard by RT-qPCR assay requires simultaneous detection of two different genes (e.g. N, Orf, and/or E) of SARS-CoV-2. In this assay, only one gene was targeted. The authors should discuss this point. 7. Did the authors perform the extraction-free RT-LAMP assay?
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Response to reviewers Reviewer #1 (Comments for the Author): (1) First, separate RNA extrac on is required before RT-LAMP (may take 1 hour manually or using equipment for many samples), so it is not "single-pot" strictly speaking; measurement of FAM/TEX615 signal needs special bulky equipment (also takes me to measure). In the end, it may not save much me compared to qRT-PCR.
"Single-pot" in this context (line 76) refers to mul plex reac ons without physical compartmentaliza on. Other references to "single-pot" have been removed. This por on of our work does not cover our proposed pla orm instrumenta on, for more on this topic see Panpradist (14). We have emphasized this manuscripts rela onship to its companion publica on to clarify focused scope of this work. See: "This effort serves as the molecular assay basis for our development of a POC diagnostic platform for SARS-COV-2 (14)." (Lines 90-91) "Sampling, storage, and portable device solutions with additional clinical and experimental evaluation are already under development (14)." (Lines 390-391) (2) LAMP-based SARS-CoV-2 assay has benefit of portability and point-of-care poten al, but this design seems only feasible for use in big labs.
The methodology, as presented here, would require a well-developed laboratory infrastructure. We believe it is readily adaptable to this environment, as its requirements are not specific to any par cular real-me amplifica on pla orm. We agree that POC applica ons are the areas of highest poten al impact, and this shared percep on is influencing our current and prior work. See: "While this proposed chemistry is amenable to adaptation to most contemporary high throughput NAAT platforms as is, it is probably best leveraged in a mobile low resource platform. Sampling, storage, and portable device solutions with additional clinical and experimental evaluation are already under development (14)." (Lines 388-391) (3) How about the cost of this LAMP (seems not low)?
As presented here, the assay chemistry itself has a cost of about $5.50/rxn with research scale ordering, however, cost is not a topic of this manuscript. For a thorough breakdown of materials costs, see Panpradist (14).
(4) Second, it looks like this RT-LAMP measures end-point fluorescence or same as qRT-PCR with dynamic curve; if similar to PCR, can Ct value be determined for RT-LAMP assay? RFU cut off is not clear for RT-LAMP, what level considered as nega ve (below 50)?
The analysis for this assay uses the described real-me parameters for the CFX so ware. This informa on has been relocated from supplemental informa on to the main text, with addi onal edits. We do not advocate using threshold me of isothermal methods as a means of sample pathogen load es ma on. See: "Fluorescence measurements for FAM and TEX 615 signal, indicating SARS-CoV-2 and IAC amplification, respectively, were taken every 25 seconds (accounting for 13 second cycle and read times). Analysis of the first 40 minutes (100 cycles) of each run was performed with Bio-Rad CFX Maestro 1.1 software (version 4.1.2433.1219) with FAM channel baseline set as 2-35 cycles and a manual threshold of 50 RFU, and Texas Red channel baseline set as 20-60 cycles with a manual threshold of 50 RFU." (Lines 154-159) (5) Third, limit of detec on is not done properly, probit analysis should be applied if this assay wants to be used in clinical labs. In Fig. 2, more replicates and dilu ons are needed (using 3 genes mixed or intact viral RNA) in order to derive LOD at 95%, at least 10 replicates (best 22) with dilu ons of RNA, 200, 100, 50, 20, 10, 5, 2.5, 1 and 0. If authors use 96-well plate (high-throughput), this can be done in one plate.
We have clarified the intent of this manuscript, which does not represent a complete end-to-end diagnos c. See: "This effort serves as the molecular assay basis for our development of a POC diagnostic platform for SARS-COV-2 (14)" (Lines 90-91) "Sampling, storage, and portable device solutions with additional clinical and experimental evaluation are already under development (14)." (Lines 390-391) (6) Actually authors did not men on if this LAMP can be done in high-throughput plate fashion.
We have added a brief statement to address this comment. See: "While this proposed chemistry is amenable to adaptation to most contemporary high throughput NAAT platforms as is, it is probably best leveraged in a mobile low resource platform." (lines 388-390) (7) Fourth, LAMP has cross-contamina on issue to address, not clear how experiments were done in Fig  3b. Authors should test samples on same plate with some high viral ter and nega ves (processed at same me from extrac on, ideally blinded), rather than separately tested. More posi ve samples are needed for robust valida on of performance (current 21).
Further clinical tes ng is performed in our companion publica on (14). We have further clarified that this is not our proposed clinical implementa on, but rather work towards that goal. Refer to responses for ques ons # 2, 5, 6.
(8) Lastly, IAC (as nega ve control) is useful, however the system lacks a sample matrix control like RP, so no way to know if NP sample is low quality; this poten ally leads to false nega ves since RT-LAMP may report as nega ve due to sub-op mal sample (but LAMP has no quality control for this). Is there any RPfailed samples (by PCR) for tes ng?
This is true, we have included language rela ng to an endogenous control in our discussion of future work. Note that RnaseP itself is a poor indicator of sample quality and is thus insufficient itself (See "DNA Cross-Reac vity of the CDC-Specified SARS-CoV-2 Specimen Control Leads to Poten al for False Nega ves and Underrepor ng of Viral Infec on", Rosebrock, 2021). See: "With the current ssDNA IAC design sampling efficacy and RNA integrity are not assessed and are essential functions for a complete NAAT diagnostic control system. Future iterations will address this by implementing an endogenous human control target and/or encapsidated RNA, such as MS2 coliphage for this purpose." (Lines 385-388) Minor comments: 1M. I wonder if some muta ons in N gene such as posi on 28881 may affect this assay performance We added in addi onal experimental data rela ng to the tolerance of the system to known mismatch muta ons present in SARS-COV-2 variants. This did not assess 29991 specifically, as that muta on does not interact with the designs presented here.

See: "Performance against variant sequences with known mismatch mutations
To practically assess the viability of multiplexed target redundancy as a strategy to mitigate diagnostic failure due to primer-template mismatches as a result of viral mutation, the sub-assays and mRT-LAMP were challenged with templates known to have mutations in target regions. Synthetic RNA templates representative of SARS-COV-2 variants Delta (Twist Bioscience, South San Francisco, California, 104539) and Omicron (Twist Bioscience, 105204), as well as the reference genome (Twist Bioscience, 102024) (RefSeq) were selected. Delta and Omicron were chosen because of their relative importance to public health (29) and the presence of fixed mismatched mutations in the NC3 and NC1 sub-assays (table 2), respectively. Sequence alignments with Delta sequences identified a high frequency single nucleotide polymorphism (SNP) mutation in that lineage (G29402T) within the NC3 B3 primer binding site Alignments with Omicron revealed two mismatches in the NC1 assay: a SNP (C28311T) in the F2 primer binding site and a 9-base deletion (GAGAACGCA28362---------) in the NC1 primer binding site. Excepting those conflicts, all other assays were perfect identity matches across their primer binding regions. RT-LAMP sub-assays were evaluated individually against these 200 copies of each template (see Supplemental methods). The NC1 sub-assay failed to detect the Omicron template, while all other sub assays and multiplex assays were successful at detection of all three templates, including Omicron (table 3)." (Lines 283-299)

See:
Added Table 3 (line 301) See: "This was further reinforced by directly challenging each of the sub-assays and the multiplex with synthetic RNA templates representative of the reference, Delta, and Omicron lineages. The Delta template was detected by all sub-assays and multiplex assays despite a mismatch in the NC3 B3 primer binding region. This mismatch affected a "bumper" primer less critical to the amplification process, and occurred internally, so tolerance to this low-risk mutation was not unexpected. By contrast, a mutation of the Omicron variant is very high risk; the 9-base deletion to the critical F1 primer binding site would be expected to completely disrupt the loop forming process essential to LAMP. As expected, the NC1 sub-assay failed to detect the Omicron template when used alone. However, the multiplexed assay was unaffected in its ability to detect Omicron." (Lines 362-370) 2M. The sensi vity should be reported as 90% in Abstract, not 100%; specificity of 100% is a bit suspicious, as UDP may have non-specificity. Maybe authors did all nega ves together (if in this case), they should validate cross-contamina on in plate format, mimicking clinical tes ng (in blind, high ter could be processed with low ter or nega ve samples together).
A valid point, we have added the sensi vity of 87% to the abstract. Our data on the specificity of this chemistry stands as reported and is not intended to be an evalua on of systemic pla orm or user specific cross-contamina on risk. See prior responses rela ng to implementa on in a clinical se ng.
See: "When challenged with extracted human samples the multiplexed assay showed 87% or better sensitivity, with 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity." (Lines 29-31) 3M. Line 53. "..diversity has rendered some NAATs suscep ble to false nega ve results,", can authors explain to readers why gene c diversity lead to false nega ves, maybe due to muta ons in the target region of an assay, is it more suscep ble for single gene target than mul ple target design; give some examples of commercial assay affected by muta ons (cita ons), e.g. S-drop out by B.1.1.7 using TaqPath.
We have added further language regarding this mechanism in the manuscript, including some specific discussion of a muta on found to affect sub-assay NC1. References (4, 5, 6, 7, others) describe this risk.

See:
"The emergence of this genetic diversity has rendered some NAATs susceptible to false negative results as a consequence of mismatches between their primers and mutations in the targeted nucleic acid, causing these tests to be altered or withdrawn by the U.S. FDA (4)." (Lines 52-55) "By contrast, a mutation of the Omicron variant is very high risk; the 9-base deletion to the critical F1 primer binding site would be expected to completely disrupt the loop forming process essential to LAMP." (Lines 366-368) 4M. Line 117. What is synthe c dsDNA gBlocks, N gene only, specify.

Edit made.
See: "All designs were tested individually and multiplexed in combination against synthetic dsDNA gBlocks ™ N gene fragment target and ssDNA IAC Ultramer ™ fragments to inform iterative design changes to individual assays." (Lines 123-125) 5M. Line 126. Give details of OpenArray, is it Taqman Real me PCR, which commercial qPCR kit was used or in-house developed. Also add columns of Ct value of N1 and N2 in Table S1.

Edits made, including a reference to the original characteriza on (21).
See: "These specimens collected from nasal or nasopharyngeal swabs were suspended in 3mL viral transport medium (Becton Dickinson 220220), aliquoted, and stored at −80°C until testing as described (21). The panel was originally characterized by TaqMan real-time PCR OpenArray plate (ThermoFisher Scientific, Waltham, MA, USA) (22) to contain at least…." (lines 128-131)
We have modified Figure 3 to clarify that it is ordinal data ("Posi ve" and "Nega ve" categories with copy number data for Posi ve vs. "Detected" and "Not Detected" categories with Threshold me data for detected IAC and target). The accompanying figure cap on has also been revised, See: Figure 3 and its cap on " Figure 3: mRT-LAMP amplification of extracted nasal specimens. Samples confirmed as Negative or Positive for SARS-CoV-2 by RT-PCR panel (N1, N2, RP) were amplified by duplicate mRT-LAMP reactions. mRT-LAMP signals for SARS-CoV-2 are shown in blue, and IAC signals are shown in orange with detected Threshold time (Tt) or "Not Detected" reported for both in all reactions; replicate pairs for each signal are connected by a line segment. Mean copy number was derived from qPCR results of N1, N2 PCR (see Supplemental Table S1)."  Reviewer #2 (Comments for the Author): 1. Whether the on-going genera on of various variants of SARS-CoV-2 affects diagnos c tests by RT-qPCR and/or RT-LAMP assays should be evaluated (or discussed). Are there any data or literatures to support this view?
We agree that addi onal informa on was needed on this topic. We added in addi onal experimental data rela ng to the tolerance of the system to known mismatch muta ons present in SARS-COV-2 variants.

See: "Performance against variant sequences with known mismatch muta ons
To prac cally assess the viability of mul plexed target redundancy as a strategy to mi gate diagnos c failure due to primer-template mismatches as a result of viral muta on, the sub-assays and mRT-LAMP were challenged with templates known to have muta ons in target regions. Synthe c RNA templates representa ve of SARS-COV-2 variants Delta (Twist Bioscience, South San Francisco, California, 104539) and Omicron (Twist Bioscience, 105204), as well as the reference genome (Twist Bioscience, 102024) (RefSeq) were selected. Delta and Omicron were chosen because of their rela ve importance to public health (29) and the presence of fixed mismatched muta ons in the NC3 and NC1 sub-assays (table 2), respec vely. Sequence alignments with Delta sequences iden fied a high frequency single nucleo de point muta on (SNP) muta on in that lineage (G29402T) within the NC3 B3 primer binding site Alignments with Omicron revealed two mismatches in the NC1 assay: a SNP (C28311T) in the F2 primer binding site and a 9-base dele on (GAGAACGCA28362---------) in the NC1 primer binding site. Excep ng those conflicts, all other assays were perfect iden ty matches across their primer binding regions. RT-LAMP sub-assays were evaluated individually against these 200 copies of each template (see Supplemental methods). The NC1 sub-assay failed to detect the Omicron template, while all other subassays and mul plex assays were successful at detec on of all three templates, including Omicron (table  3)." (Lines 283-299) See: Added Table 3 (line 301) See: "This was further reinforced by directly challenging each of the sub-assays and the mul plex with synthe c RNA templates representa ve of the reference, Delta, and Omicron lineages. The Delta template was detected by all sub-assays and mul plex assays despite a mismatch in the NC3 B3 primer binding region. This mismatch affected a "bumper" primer less cri cal to the amplifica on process, and occurred internally, so tolerance to this low-risk muta on was not unexpected. By contrast, a muta on of the Omicron variant is very high risk; the 9-base dele on to the cri cal F1 primer binding site would be expected to completely disrupt the loop forming process essen al to LAMP. As expected, the NC1 sub-assay failed to detect the Omicron template when used alone. However, the mul plexed assay was unaffected in its ability to detect Omicron." (Lines 362-370) 2. Mul plex RT-LAMP system based on displacement FIP/BIP probes have been developed (BioTechniques 2012, 53:81-89;Anal. Chem. 2016, 88, 3562−3568). In this study, the authors using universal displacement probe linking/binding to loop primer. In fact, this strategy (based on loop primers) was not novel (had been published previously (Anal. Chem. 2018, 90, 4741−4748;PLoS ONE 2021, 16(3): e0248042: this paper focused on SARS-CoV-2 detec on). The authors did not men on the previous papers. On the other hand, the principle is similar between the two strategies (based on FIP/BIP and FL/BL primers). The authors should men on and discuss (compare) the advantages and disadvantages of both methods.
See: "A variety of fluorescence probes systems have been previously described (13) and this remains an ongoing area of LAMP innovation." (Lines 83-84) "The UDP probe system that enables differentiable detection of Target and IAC amplifications has many commonalities with existing probe systems, but has a unique feature set useful for rapid development, pooled target amplification reporting, and flexible application. Many of the previously described probe systems, such as DARQ (10) and Molecular Beacons (32) are specific to endogenous target sequence and require dedicated probes. This is also true of assimilating probes (33), the technology that is conceptually most similar to UDPs. UDPs leverage a similar design, exploiting the existing compatible functionality of the loop primers, but use an adapter intermediate so that probe sequences are not directly tied to the target sequence and can be entirely engineered. This provides several advantages: the probes can be designed to be minimally interactive with other elements of the amplification mix, they can be repurposed or adapted to new or revised designs without the need to develop a new probe, and in a multiplex reaction multiple targets can be efficiently associated with a single reporter probe. Mediator displacement probes (34) share these properties, but do not incorporate the probe label into the amplicon which may limit some applications, requires an additional Mediator oligonucleotide, and has a more complex dual labeled stemloop probe structure. While each of the previously described probe systems has been shown to be effective in various contexts, the suitability of UDPs to a multiplexed target redundant system for a rapidly mutating and variable target with an IAC is apparent." (Lines 327-342) 3. Mismatch/variant-tolerant LAMP method had been developed previously for efficient detec on of highly variable RNA viruses (e.g. HIV-1) (Front Microbiol 2019，10:1056Analyst 2021, 146: 5347-5356), and showed well performance. The authors should men on and discuss (compare) the advantages and disadvantages of two kinds of strategies for detec on of highly variable viruses/targets. This method was also used for detec on of SARS-CoV-2 (Virologica Sinica，2020, 35:344-347).
We originally cited an earlier paper by the same lead author (Ref#37). We have included some commentary about its compa bility, as well as highlighted a specific example where a mismatch tolerant method would fail.
See: "While altering or updating a single target assay in a relevant timescale to address Omicron would be logistically challenging, and mismatch tolerant LAMP methods (37) would still be vulnerable to such a large deletion, a multitarget assay is robust to the threat. In this scenario, the affected primers could be redesigned to be inclusive to the variant, potentially including other mismatch tolerance strategies, and phased into the multiplexed assay without disruption to the diagnostic and ensuring no lapse in coverage over time." (Lines 372-377) 4. The performance of this mul plex RT-LAMP assay on various mismatches between primers and templates should be evaluated like previous studies (Front Microbiol 2019，10:1056Analyst 2021, 146: 5347-5356).
See response to reviewer 2 ques on 1.
5. In the clinical evalua on experiments, 30 SARS-CoV-2-pos ve samples were used. First, the ample size was too small. Second, did these samples contain some SARS-CoV-2 variants? Third, clinical samples used in the evalua on should contain at least two-three most common variant. If there is some difficulty in the collec on of clinical samples carrying variants, simulated samples with in vitro synthesized/transcribed variants' RNA should be used.
See response to reviewer 2 ques on 1.
See: "Sampling, storage, and portable device solutions with additional clinical and experimental evaluation are already under development (14)." (Lines 390-391) 6. In addi on, the gold standard by RT-qPCR assay requires simultaneous detec on of two different genes (e.g. N, Orf, and/or E) of SARS-CoV-2. In this assay, only one gene was targeted. The authors should discuss this point.
The gold standard tes ng employed here was based on the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnos c Panel consis ng of two COV targets, both of which are located in the N gene. In this test, both the N1 and N2 targets require a posi ve to call a posi ve, but no formal explana on is provided for this requirement in official guidance. We have refrained from commen ng on this requirement but adhered to it in our reference tes ng. This is no longer considered the standard of prac ce following updates to the EUA CDC approved diagnos c for SARS-COV-2.
7. Did the authors perform the extrac on-free RT-LAMP assay?
No, we did not. We have addressed this comment and this limita on of the study in the text by direc ng the reader to the relevant study.
See: "The preliminary testing performed suggests that the system is tolerant to inhibitors that might typically interfere with a direct-to-amplification workflow. However, the clinical specimens evaluated here were processed by RNA extraction. In-amplification sample lysis, and testing with human sample matrix is necessary to further validate this strategy. Assessment of direct detection of contrived samples in nasal matrix and appropriate crude clinical samples is a crucial aspect of ongoing work and was explored in our companion publication (14) focused on a POC detection platform." (Lines 378-384) Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
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Editor, Microbiology Spectrum Reviewer (Editor) comments: Please modify Abstract to include specific numbers of human samples used in determining the sensitivity and the specificity.

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