Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples

Dawn M Dudley; Christina M Newman; Andrea M Weiler; Mitchell D Ramuta; Cecilia G Shortreed; Anna S Heffron; Molly A Accola; William M Rehrauer; Thomas C Friedrich; David H O’Connor

doi:10.1371/journal.pone.0244882

. 2020 Dec 31;15(12):e0244882. doi: 10.1371/journal.pone.0244882

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples

Dawn M Dudley ¹, Christina M Newman ¹, Andrea M Weiler ², Mitchell D Ramuta ¹, Cecilia G Shortreed ¹, Anna S Heffron ¹, Molly A Accola ³, William M Rehrauer ³, Thomas C Friedrich ⁴, David H O’Connor ^1,^*

Editor: Ruslan Kalendar⁵

PMCID: PMC7775089 PMID: 33382861

Abstract

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/μl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

Introduction

There are more than 13.8 million reported severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in the United States as of December 3rd, 2020 (https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/cases-in-us.html). The actual number of infections is likely far greater since testing remains limited. Asymptomatic individuals contain similar levels of SARS-CoV-2 in the upper respiratory tract as symptomatic individuals [1–6]. Furthermore, 17 out of 24 (71%) presymptomatic patients had positive viral cultures 1 to 6 days before the onset of symptoms [1]. Symptom-based testing is not sufficient for controlling SARS-CoV-2 transmission and emphasizes the need for expanded nucleic acid surveillance of asymptomatic/presymptomatic individuals.

Conventional SARS-CoV-2 testing relies on RT-PCR amplification of virus-specific nucleic acids extracted from nasopharyngeal (NP) swabs. However, shortages of nucleic acid extraction and RT-PCR reagents as well as RT-PCR instrumentation, remain a problem [7]. In addition, this test is expensive ($25/sample for reagents) and turn-around time is often several days [8–10]. Alternative nucleic acid extraction methods and "direct" testing that does not require nucleic acid extraction are important to expand testing while reducing time and cost. For example, the SalivaDirect method recently approved under an FDA EUA utilizes saliva without RNA extraction into an RT-PCR assay [11].

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been for point-of-need diagnostic testing for several pathogens, including SARS-CoV-2 [12–21]. RT-LAMP assays are an alternative method for rapidly detecting the presence of specific nucleic acids in samples, with colorimetric or fluorescent visualization of results. RT-LAMP assays are inexpensive (~$7/sample), high-throughput (can be run in a 96-well format), do not necessarily require nucleic acid purification, and give rapid results (~60–90 minutes from set-up to results). Previously published manuscripts demonstrate proof-of-principle for SARS-CoV-2 testing by RT-LAMP using either contrived samples with free nucleic acid or extracted RNA from primary samples [12–14,16–22]. Minimally processed primary NP swab samples are more challenging, since biological inhibitors such as nucleases may hinder amplification or degrade RNA. Here we describe the LOD of direct RT-LAMP without RNA isolation on primary NP swab samples.

Direct RT-LAMP is an example of a lower sensitivity, fast-turnaround test that requires minimal equipment that can be used in a point-of-need setting. SARS-CoV-2 antigen tests provide a similar point-of-need, lower sensitivity, and rapid test. The CDC recently released guidance highlighting the importance of point-of-need antigen testing for screening asymptomatic individuals without known SARS-CoV-2 exposure [23]. These tests are also in limited supply. The importance of quick-turnaround and inexpensive tests is becoming increasingly recognized to help mitigate transmission of SARS-CoV-2 [24–26]. The current gold-standard qRT-PCR test has proven to have a turn-around time of several days and therefore does little to move highly contagious individuals into isolation before they transmit the virus [25]. Analytical modeling of different screening strategies shows that very frequent, inexpensive, and even poorly sensitive testing is predicted to sufficiently isolate positive individuals and prevent widespread transmission better than low frequency highly sensitive testing [27]. Direct RT-LAMP, therefore, has the potential to become an important addition to the currently available SARS-CoV-2 testing arsenal.

In this study, we focused on characterizing and optimizing direct RT-LAMP without RNA isolation and with primary NP swab samples with known SARS-CoV-2 status. We demonstrate the limit of detection (LOD) of direct swab RT-LAMP in primary swab samples as well as modifications that help improve sensitivity but don’t rely on the same materials required for traditional qRT-PCR methods. We characterized the use of Lucigen QuickExtract (QE) lysis buffer, guanidine hydrochloride addition, an alternative RNA isolation method, and several primer sets and combinations targeting different gene regions. Systemic evaluation of these modifications with primary samples will be useful to other groups designing RT-LAMP workflows for SARS-CoV-2 surveillance.

Materials and methods

Sample collection

Residual, completely de-identified NP swab samples were provided by the University of Wisconsin-Madison Hospitals and Clinics (UWHC) and the Wisconsin State Laboratory of Hygiene (WSLH) under biosafety protocol B00000117 (IRB 2016–0605), and their use was not considered human subjects research by the University of Wisconsin-Madison School of Medicine and Public Health’s Institutional Review Board. Samples were not specifically collected for this study. Samples were collected into a variety of transport media including universal transport media (UTM), viral transport media (VTM), and phosphate-buffered saline (PBS), stored at 4°C for up to 7 days, and transported to the laboratory at room temperature. Upon arrival at the laboratory, samples were stored at either 4°C (for immediate same-day use) or -80°C until use in RT-PCR or RT-LAMP assays.

qRT-PCR

Viral load analysis was performed after samples arrived in our laboratory. RNA was isolated using the Viral Total Nucleic Acid kit for the Maxwell RSC instrument (Promega, Madison, WI) following the manufacturer’s instructions. Viral load quantification was performed using a qRT-PCR assay developed by the CDC to detect SARS-CoV-2 (specifically the N1 assay) and commercially available from IDT (IDT cat # 10006770) (Coralville, IA) [28]. The assay was run on a LightCycler 96 or LC480 instrument (Roche, Indianapolis, IN) using the Taqman Fast Virus 1-step Master Mix enzyme (Thermo Fisher, Waltham, MA). The LOD of this assay is estimated to be 200 genome equivalents/ml in swab media. To determine the viral load, samples were interpolated onto a standard curve consisting of serial 10-fold dilutions of in vitro transcribed SARS-CoV-2 N gene RNA kindly provided by Nathan Grubaugh (Yale University).

RT-LAMP

The experiments we describe here were modified from the SARS-CoV-2 RT-LAMP assay developed by Zhang et al. [13]. We used fluorescent-based detection with Warmstart LAMP reagents and the included fluorescent dye (New England Biolabs, NEB cat # E1700L). We tested primer sets developed in previous studies targeting several SARS-CoV-2 genes as shown in S1 Table [13,14,22,29–33]. Of note, the Color-Orf1a primers and Lamb-Orf1a primers are identical but were used at different concentrations per the protocols developed by each originating lab. The final 1X primer concentrations are listed in S1 Table. For each reaction, a 10X stock of all 6 primers was combined with Warmstart mastermix and water in 25μl reactions following the manufacturer’s recommendations. Unless otherwise stated, 1μl RNA transcript of the SARS-CoV-2 N-gene obtained by Dr. Nathan Grubaugh (S2 Table), 1μl of synthetic SARS-CoV-2 RNA transcript (Twist Biosciences; RNA control 2), 1μl of gamma-irradiated SARS-CoV-2 (BEI; NR-52287; isolate USA-WA1/2020), or 1μl primary NP swab sample were tested in each RT-LAMP reaction. Unless otherwise stated, all serial dilutions were performed in water. For reactions testing guanidine hydrochloride addition to the RT-LAMP mastermix, a final concentration of 40mM stock was used in the mastermix. Except where otherwise specified, samples were run on a Roche Lightcycler 96 instrument (Roche Diagnostics) using an 80-cycle program with the SYBR Green channel at 65°C (495–497 nm absorption; 517–520 nm emission) with each cycle representing data collected every 30 seconds. The presented Cq value represents the cycle number where detection of the RT-LAMP amplicon begins. The more template in the reaction and the more efficient the reaction conditions are, the earlier the detection begins. For experiments determining the appropriate volume of direct swab sample addition for highest RT-LAMP efficiency, a 60-cycle program with data collection every 20 seconds was used.

The specificity of the primers tested in this manuscript were established in previous publications [13,14,22,29,31–33]. Briefly, the Gene-N-A primers, used in most of the experiments in this manuscript, did not cross-react with SARS-CoV-1 N-gene by RT-LAMP and were not anticipated to cross-react with other common respiratory pathogens based on sequence comparisons (communication with Nathan Tanner). The Gene-N2, Gene-E1, and As1e primers were aligned against other coronaviruses and respiratory pathogens and the only pathogen with >80% identity to SARS-CoV-2 in more than one of the 6 primers required for RT-LAMP was SARS-CoV-1 (communication with Nathan Tanner and Brad Langhorst), which hasn’t circulated in humans since 2004. The Color Genomics primers were tested for cross-reactivity against 51 organisms for EUA approval and were not cross-reactive to any organism tested [33].

Sample lysis

A subset of samples were treated with LucigenQE RNA Extraction Solution (Lucigen, Middleton, WI) in a 1:1 ratio as described in Ladha et al. [34]. Briefly, NP swab media was combined with an equivalent volume of LucigenQE and briefly vortexed. Samples were then incubated for 5 minutes at 95°C, cooled on ice, and maintained until the addition to the RT-LAMP reaction.

Statistical analysis

To assess improvement in quantification cycle (Cq) values using sample lysis with LucigenQE or RNA isolation, mean Cq values were calculated for each sample. Mean Cq values were not normally distributed for either dataset so we used a nonparametric equivalent to a paired t-test, the Wilcoxon signed rank test with continuity correction, for each set of paired samples.

To examine whether sample vRNA load and/or treatment were significantly associated with a positive RT-LAMP result, we used logistic regression with the RT-LAMP result as the dependent variable. For our analysis, an equivocal result in which one replicate was positive while the other was negative, was conservatively treated as negative. We coded RT-LAMP results for each sample tested by each method as a dichotomous outcome with positive samples coded as “1” and negative or equivocal samples coded as “0”. For explanatory variables, we chose qRT-PCR vRNA load, with samples greater than 10³ copies/μl coded as “1” and samples less than 10³ copies/μl coded as “0”, and group, designated as either 5μl RNA, 1μl lysed, or 1μl direct addition.

All statistical analyses were performed in RStudio (v. 1.2.1335) using R (v. 3.6.0) [35].

Results

Limit of detection with RNA transcript and Gene-N-A primers

To determine a limit of detection for the RT-LAMP assay, serial 10-fold dilutions of RNA transcripts containing the N-gene were tested in RT-LAMP reactions with Gene-N-A primers in duplicate in 3 independent assays. RNA transcripts were diluted in RNase-free water. Consistent detection of RNA was achieved when 1x10³ copies or greater of RNA/μl was added into the reactions (Fig 1A). To obtain a more precise LOD, the transcript was diluted 1:2 starting at 5x10³ copies/μl down to 78 copies/μl and each concentration was run in 10 replicates. Nine of ten replicates at 625 copies/μl were positive, while 6/10 were positive at 312 copies/μl and 5/10 were positive at 156 copies/μl (Fig 1A). Thus, we can consistently detect 625 copies of input into the reaction but can detect down to 156 copies of input in half of the reactions. Zero reactions were detected as positive at 100 copies/μl or below.

Fig 1 — (A) The quantification cycle (Cq) relative to each transcript copy number is plotted. Samples that were not detectable were plotted on the line labeled ND for all graphs at Cq of 60 or 80, equal to the total number of cycles. The vertical line is set at the lowest dilution where positive samples were detected using the transcript input for all graphs (156 vRNA copies/μl). Each replicate is plotted on all graphs. (B) Detection of 106 SARS-CoV-2 positive primary NP swab samples relative to their in-house viral load value. (C) RT-LAMP Cq of five SARS-CoV-2 positive primary NP swab samples with different swab input volumes.

Limit of detection with primary nasopharyngeal swab samples

Residual NP swab samples from 106 patients with diagnosed SARS-CoV-2 and 31 negative samples were tested directly by RT-LAMP in duplicate. Additionally, RNA was isolated from the positive samples and tested by qRT-PCR with a transcript standard for quantitation. A total of 63/106 (59%) samples tested positive by RT-LAMP and 106/106 by qRT-PCR (S3 Table). Another 13 samples were equivocal by RT-LAMP, with one of two replicates positive. As shown in Fig 1B, the LOD of primary samples was similar to that seen with the RNA transcript. For the rest of the analysis, we focus on the ability of this assay to detect samples with a viral load of > 1x10³ copies/μl as a conservative LOD based on our transcript LOD of 625 copies/μl. 63/78 (81%) samples with viral RNA copy numbers greater than 1x10³ copies/μl were detected by RT-LAMP, whereas 0/28 samples with concentrations <1x10³ copies/μl were detected positive and 3/28 were equivocal. All 31 samples that tested negative for SARS-CoV-2 by clinical laboratories also tested negative by RT-LAMP (S3 Table samples 107–137). Negative samples were not tested by our internal qRT-PCR assay. Five of the 31 samples that tested negative for SARS-CoV-2 tested positive for other respiratory pathogens including influenza A, rhinovirus, respiratory syncytial virus, influenza B, and human metapneumovirus. In this limited sample set, the Gene-N-A primers showed 100% specificity for SARS-CoV-2.

RT-LAMP is inhibited by adding larger volumes of primary sample

To determine whether adding larger volumes of primary NP swab samples could improve the sensitivity of the assay, 1, 3, and 5μl of swab samples from 5 primary SARS-CoV-2-positive samples were tested side-by-side. All replicates were detected as positive when 1μl was added directly into the RT-LAMP reaction (Fig 1C). However, one of the two replicates from two samples tested negative when 3μl of the sample was added and both replicates from one sample tested negative when 5μl of the sample was added into the RT-LAMP reaction. Furthermore, Cq thresholds were higher with the addition of higher volumes of sample. Therefore, we chose to use 1μl of straight swab sample in subsequent experiments.

Lysis buffer improves the sensitivity of RT-LAMP

To determine whether treatment with lysis buffer improves the sensitivity of RT-LAMP to detect SARS-CoV-2 in clinical samples without the use of traditional nucleic acid isolation methods, we treated 72 clinical samples with a range of SARS-CoV-2 vRNA loads in a 1:1 ratio with LucigenQE as described by Ladha et al. [34]. We then compared the fluorescent RT-LAMP Cq values between 1μl of lysed sample and 1μl of the same samples added directly. The addition of 1μl of NP swab eluate directly into the RT-LAMP reaction resulted in positive detection in both replicates of 46/72 (64%) known SARS-CoV-2-positive samples and in 1 of 2 replicates in 6 additional samples (Fig 2A, S3 Table). Treatment of the same 72 samples with LucigenQE resulted in detection of both replicates for 56/72 (78%) samples, an additional 10 samples that were undetectable before. An additional 4 samples that were negative when tested directly were equivocal when treated with LucigenQE. Focusing on samples above 1x10³ copies/μl, 45/53 (85%) samples were positive without extraction while 53/53 (100%) were positive with LucigenQE extraction. Without RNA extraction an additional 5 samples were equivocal indicating that extraction resulted in more consistent detection of positive samples in both replicates above the 1x10³ copies/μl threshold. While 0/19 samples with viral loads below 1x10³ copies/μl were positive with 1μl straight, 4 of 19 samples were detectable after lysis in both replicates and 4 additional samples were detected in 1 of 2 replicates. Mean Cq for LucigenQE-treated samples were significantly lower (Cq = 38.62) than those for directly added samples (Cq = 49.08) (Wilcoxon signed rank test, V = 1830, p = 1.67E^-11), suggesting that lysis treatment improves the efficiency of amplification in the LAMP reaction (Fig 2A). We also examined whether sample vRNA load and/or treatment were significantly associated with RT-LAMP detection results. We found that direct addition of 1μl of untreated NP swab was associated with a decreased odds of detecting a positive RT-LAMP result (OR = 0.20, 95%CI = 0.04–0.65, p = 0.015) while the most important factor associated with a positive result was a sample vRNA load of greater than 1x10³ copies/μl (OR = 88.5, 95%CI = 25.74–434.87, p = 1.88E^-10).

Fig 2 — (A) Comparison of Cq values between samples treated with or without LucigenQE and run by RT-LAMP with 1μl of sample. (B) Comparison of Cq values between RT-LAMP assays run with 1μl straight swab or 5μl of isolated and purified vRNA. vRNA copies/μl are reported for the starting sample concentration before RNA purification and concentration.

RNA isolation improves the limit of detection of RT-LAMP to levels similar to qRT-PCR

One of the primary reasons the direct RT-LAMP assay is less sensitive than the diagnostic qRT-PCR assay is because the qRT-PCR assay uses 5μl of concentrated and purified RNA as input. To determine whether the RT-LAMP assay would perform to a similar level of detection if the same input was used, 5μl of purified RNA was used in RT-LAMP assays from a subset (n = 44) of COVID-19-positive NP swab samples. The starting sample viral loads ranged from 1.01x10¹ to 1.14x10⁷ copies/μl. 25 of 44 had concentrations of virus above the RT-LAMP LOD of 1x10³ vRNA copies/μl. Of the 44 samples tested with 1μl of the direct swab, 18 tested positive (43%) and 7 were equivocal between replicates (Fig 2B and S3 Table). When 5μl of purified RNA was used in the reactions instead, 42 of 44 samples (95%) tested positive (Fig 2B, S3 Table). Note that the process of RNA isolation used in this experiment concentrated the RNA approximately 3-fold from the concentration presented in Fig 2B and that 5μl of that concentrated RNA was used, while 1 μl of the starting sample concentration was used in the 1μl straight reactions. Samples with 100-fold lower vRNA copies/μl were detected after RNA isolation and concentration by RT-LAMP. Adding purified RNA brings the possible LOD of detection down to 150 copies of input of a 10 copies/μl sample, which was detected in 4 of 6 primary samples tested within this viral load range. This is similar to the detection of 156 copies of transcript as shown in Fig 1A. Of the 25 samples with viral loads over the 1x10³ copies/μl threshold, 18 (72%) were positive when adding straight while 25/25 (100%) were positive with RNA isolation.

Overall, the direct addition of 1μl NP swab was associated with reduced odds of a positive RT-LAMP result (OR = 0.0054, 95%CI = 0.00023–0.041, p = 2.56E^-05). Similar to the results for lysis buffer treatment, samples with qRT-PCR vRNA loads greater than 1x10³ copies/μl had significantly increased odds of a positive RT-LAMP result (OR = 49.35, 95%CI = 8.22–966.29, p = 0.00044). Lastly, the Cq values were lower for all detected samples when 5μl of RNA was added (mean Cq = 31.42) instead of 1μl straight sample (mean Cq = 58.72), indicating faster and more robust detection with concentrated and purified RNA (Wilcoxon signed rank test, V = 903, p = 1.71E^-08) (Fig 2B).

Alternative primers improve efficiency

Multiple SARS-CoV-2 RT-LAMP primer sets have been published or included in manuscripts on preprint servers since we began our experiments. We compared the efficiency of several alternative primer sets either alone or in combination with the Gene-N-A primers used in our initial studies (S1 Table). Two primary NP swab samples with high concentrations of SARS-CoV-2 (NP1:1.09x10⁹ copies/μl, NP2:4.28x10⁷ copies/μl) as well as gamma-irradiated SARS-CoV-2 (BEI) were used to screen different primer combinations using the same fluorescent RT-LAMP conditions. First, a set of primers previously established [14,30] and used to obtain an FDA EUA by Color Genomics was tested with each primer alone and in different combinations (Fig 3A). Several primers and primer combinations resulted in a lower Cq value across the board than the Gene-N-A gene primers suggesting improved reaction efficiency. The primer combinations including Color-N/Color-E and Color-N/Color-ORF1a yielded the lowest Cq values in all samples.

Fig 3 — Reactions were run in duplicate and both replicates are shown on the graphs. Samples that were not detectable are plotted on the ND line at Cq 80. (A) Comparison of Cq values using Color Genomics primers to the Gene-N-A primers on two primary NP swab samples and irradiated SARS-CoV-2. (B) Comparison of Zhang et al. [22] primers to a subset of Color Genomics primers and Gene-N-A primers on two primary samples and irradiated SARS-CoV-2. (C) Comparison of the Cq values obtained when using the best primers and combinations of primers across different dilutions of SARS-CoV-2 TWIST RNA with and without GuHCl.

Next, we compared the Gene-N-A, Color-N, Color-E, and Color-N/E combination to second-generation primers described in Zhang et al. [22] (Gene-N2, Gene-E1 and As1e). We found that As1e, originally published by Rabe et al. [29], yielded the lowest Cq value followed closely by a combination of As1e with the two primers targeting the Gene-N2 and Gene-E1 genes designed by Zhang et al. (Fig 3B). The Color-N primer yielded similar Cq values to the triple combination primer set from Zhang et al. We also tested the Color-N, Color-E1, and Gene-N-A gene primer sets against additional published primers that target ORF1a (Lamb, Yu, El-Tholoth, and Zhang primers) either alone or in combination and compared them to the Gene-N-A gene primer set (S1 Table). The Color-N primer produced the lowest Cq value in this set (S1 Fig).

We then tested As1e, Color-N, As1e/Color-N/Gene-E1 primer set with and without guanidine hydrochloride, as recommended by Zhang et al., with Twist RNA SARS-CoV-2 template [22]. Under these conditions, guanidine hydrochloride improved detection with most primer sets with the exception of the Gene-N-A primer set (Fig 3C). Using the Twist RNA, the primer set that detected samples at the lowest dilutions was the Color-N primer or combination of As1e/Color-N/Gene-E1 with guanidine hydrochloride, though only in one of two replicates. The As1e primer with and without GuHCl as well as the combination As1e/Color-N/Gene-E1 with GuHCl often yielded the lowest Cq value.

Lastly, to determine which primer set or combination worked best with primary NP swab samples, fourteen samples with viral loads ranging from 7.33 x10⁴-1.52 x10⁸ vRNA copies/μl were tested using 1μl of straight swab sample with the most promising primer combinations with and without GuHCl. Both the Color-N primer set alone and the As1e/Color-N/Gene-E1 combination yielded the lowest Cq value across the different levels of virus (Fig 4A). Guanidine hydrochloride did not improve detection in primary samples as seen with the Twist RNA. Twelve additional primary NP swab samples with high Ct values ranging from 25 to 35 were tested with the Color-N and As1e/Color-N/Gene-E1 combination. Detection of these samples was very similar between the two primer conditions (Fig 4B). Note that several samples were equivocal at Ct 25 for both primer sets equivalently and that we might expect better detection at that threshold. Since we do not eliminate inhibitors in the samples and since these samples were stored for several days and freeze-thawed between the time the hospital ran the sample to acquire the Ct value and the time the sample was run in our assay, some of the replicates became undetectable. We would expect to more consistently detect virus at a Ct of 25 in both replicates in fresh samples.

Fig 4 — (A) Comparison of three primer sets or combinations either with or without GuHCl on 14 primary NP swab samples with high viral load copy numbers. (B) Comparison of the Color-N primer set to As1e/Color-N/Gene-E1 on primary NP swab samples with lower levels of virus. These samples were not run by our in-house viral load test and therefore Ct value obtained from the hospital is reported.

Discussion

Frequent, widespread testing is considered the best mitigation strategy to control SARS-CoV-2 before a vaccine or effective therapy becomes widely available in 2021. Traditional qRT-PCR is sensitive but is time-consuming and reliant on very specific reagents that are in short supply. RT-LAMP has become a promising alternative to qRT-PCR, but the sensitivity of this assay has been poorly characterized from primary NP swab samples when added directly into the reaction. Many published studies establish a LOD based on free RNA transcript or isolated RNA from cultured virus of around 100 copies/rxn. These LODs apply to RT-LAMP only when purified RNA is used as input. They do not apply to direct RT-LAMP methods containing whole virions in primary samples, which also contain host enzymes and other host components. In this study, we established that 625 copies/reaction was necessary in order to detect RNA transcripts consistently in 9/10 reactions in our assay. In primary samples we rarely detected virus in samples with a vRNA load of less than 5x10²-1x10³ vRNA copies/μl, establishing this threshold as a conservative LOD. While not as sensitive as methods with RNA extraction first, this LOD range is sufficient for SARS-CoV-2 surveillance to detect virus in individuals with the minimal amount of virus necessary to isolate the virus and therefore most likely to transmit the virus [1,36–38].

The LOD of 1x10³ copies/μl is likely due to inefficiencies associated with virus lysis at 65°C during the LAMP reaction and possible degradation of liberated RNA by enzymes, including RNases, present in primary samples. Indeed, adding more sample, including more host enzymes and media with potential inhibitors, reduced detection. On the other hand, RNA extraction eliminates these inhibitors and allows 100% consistent detection above 10 copies/μl (150 copies/reaction). Lysis with LucigenQE was compatible with RT-LAMP and improved our sensitivity to detect SARS-CoV-2 in primary samples with vRNA loads less than 10³ copies/μl. When compared with direct addition, we were able to increase our detection of true positives for both replicates from 61% to 78% after lysis treatment. In samples with viral loads above 1x10³ copies/μl we improved detection to 100%. Guanidine hydrochloride has also been shown to improve sensitivity in other studies and while our results showed better detection with synthetic Twist RNA, bringing the LOD down to 62.5 copies/μl, the same improvement was not observed in primary samples in our hands. The largest increase in sensitivity occurred with RNA isolation prior to RT-LAMP using an alternative RNA isolation method to those approved for the CDC qRT-PCR assay. With RNA isolation we detected 95% of the samples detected by qRT-PCR, including those with the lowest viral loads.

There are now many primers available targeting different regions of SARS-CoV-2. In this work, we tested several sets to iteratively choose which primer set worked best with primary NP swab samples. We found that several primer sets performed more efficiently than the Gene-N-A primers we used in most of our experiments. The two best performing primer sets that were nearly indistinguishable in performance with both high and low viral load primary samples were a combination of As1e/Color-N/Gene E1 and the Color-N primer set alone.

For this study, we chose to use fluorescent RT-LAMP for detection rather than colorimetric detection. Using fluorescence enabled analysis of the differences in the Cq values providing a quantitative evaluation of how each condition changed the efficiency of the assay. However, when considering how to deploy RT-LAMP in the field, our group has developed a mobile RT-LAMP workflow that uses saliva and colorimetric readouts for low cost and portability [39]. Additional work needs to be done to determine whether the benefits of fluorescent detection can be inexpensively migrated to decentralized point-of-need testing.

Many studies are comparing RT-LAMP to RT-PCR results presented as Ct value, rather than vRNA copies/μl. We chose to focus on comparing methods and determining LODs based on a standard qRT-PCR assay performed in our laboratory with a quantitative standard, rather than Ct values generated by the varying sources of our primary samples. Diagnostic laboratories have transitioned between different methods as reagents were available and as new assays became available, which means that the Ct values can vary. By comparing all our results to the vRNA copies/ml that we generated in our lab using a consistent primer set and protocol (CDC qRT-PCR assay) that targeted the same gene (and primers) as our SARS-CoV-2 RT-LAMP assay, we were able to ensure our comparisons were consistent across all samples.

Overall, we have shown that direct RT-LAMP using fluorescent detection can detect SARS-CoV-2 in primary NP swab samples with viral loads greater than 1x10³ vRNA copies/μl. We were able to improve this slightly with the quick and low-cost addition of Lucigen lysis buffer to the reaction and could detect 100% of samples above the 1x10³ copies/μl threshold. We also saw improvement in efficiency with several alternative primer sets. While direct RT-LAMP is not as sensitive as qRT-PCR, it is sufficient to detect the levels of virus that are necessary to culture virus from a sample. This means that this assay detects people who are likely to transmit the virus for a significant reduction in cost, time, and reagents relative to qRT-PCR. Direct RT-LAMP costs approximately $7 per sample while qRT-PCR costs $25. RT-LAMP from set-up to results can be performed in 60–90 minutes depending on the scale of the assay (more samples take longer to set up on the front end, but the reaction time is 30 minutes), while qRT-PCR requires 60 minutes for RNA extraction, 30 minutes to set-up qRT-PCR, and an additional 90 minutes for the qRT-PCR run to finish. One proposed way to utilize this test could be to surveil large numbers of individuals who are then directed toward diagnostic testing by qRT-PCR if they test positive by this test, significantly reducing the burden on diagnostic labs and their resources. This test, if transitioned to a colorimetric version, could be set up at point-of-need sites with minimal equipment and could provide same-day test results to individuals in a work or school environment. For this test to be used in a clinical or diagnostic capacity, additional clinical validation would be required. Overall, direct RT-LAMP is an important addition to the repertoire of currently available tests to identify samples containing SARS-CoV-2 nucleic acids.

Supporting information

S1 Fig. Comparison of Gene-N-A, Color-N, and Color-E primers to several ORF1a -targeting primer sets and combinations with two primary samples and irradiated SARS-CoV-2.

Samples that were not detectable were plotted on the ND line set at Cq 80, the hißghest cycle number in our assay.

(TIF)

Click here for additional data file.^{(479.8KB, tif)}

S1 Table. Primer sequences and final 1X concentrations used in the RT-LAMP reactions.

(DOCX)

Click here for additional data file.^{(27.2KB, docx)}

S2 Table. Nucleotide sequence of the RNA transcript provided by Dr. Nathan Grubaugh.

(DOCX)

Click here for additional data file.^{(13.4KB, docx)}

S3 Table. qRT-PCR N-gene viral loads and RT-LAMP Cq values from 106 primary NP swab samples run in duplicate with 1ul of swab sample or a subset of NP swab samples run in duplicate with either 1μl of primary samples treated with Lucigen QuickExtract or 5μl of extracted vRNA.

(DOCX)

Click here for additional data file.^{(32.1KB, docx)}

Acknowledgments

We thank Nathan Tanner for discussions about primers and optimizations of the RT-LAMP assay. We thank Brad Langhorst for specificity data for NEB primer sets. We thank Dr. Nathan Grubaugh for the Sars-CoV-2 N-gene transcript. The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, Gamma-Irradiated, NR-52287.

Data Availability

All relevant data are within the manuscript and its Supporting information files. Additional details about protocols and results are posted at https://openresearch.labkey.com/Coven/wiki-page.view?name=lamp-testing.

Funding Statement

This work was supported in part by the Office of Research Infrastructure Programs/OD under grant P51OD011106 awarded to the Wisconsin National Primate Research Center at the University of Wisconsin-Madison (awarded to DHO and TCF) and more information is available at https://orip.nih.gov/. This work was also supported by the Rapid Acceleration of Diagnostics (RADX) program through the National Institutes of Health (grant number 144 AAI2136 awarded to DHO and TCF) and more information is available at https://www.nih.gov/research-training/medical-research-initiatives/radx. Lastly, this work was also funded by the Wisconsin Alumni Research Foundation COVID19 Accelerator Challenge (grant number 135 AAH9333 awarded to DHO and TCF) and more information is available at https://www.warf.org/programs-events/community/covid-19-updates/. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0244882.r001

Decision Letter 0

Ruslan Kalendar

26 Oct 2020

PONE-D-20-29372

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab and saliva samples

PLOS ONE

Dear Dr. Dudley,

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2. Please clarify if the biological samples used in your study were:

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(2) specifically collected for this study or not

(3) whether the samples were collected through a medically prescribed test

(4) whether the samples were completely de-identified before researchers accessed the samples.

3. Please provide the catalog number for the:

a) coronavirus qRT-PCR assay used in this study and

b) 4 Warmstart LAMP reagents and the included fluorescent dye (New England Biolabs, NEB).

4. Please provide the sequence of the in vitro transcribed SARS-CoV-2 N gene RNA provided by Nathan Grubaugh.

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Reviewer #1: Thanks to the authors for this very careful work on a diagnostic system for SARS-Cov-2. Given the context of the current pandemic and recurring shortages of reagents, any serious studies evaluating the performance of diagnostic tests are important.

I have some comments on the body of the text and a general remark on the introduction and discussion of this work.

It is obvious that the system presented (without conventional prior DNA extraction) lacks sensitivity unlike the gold standards. The authors from the introduction endeavor to cite all the literature which justifies that beyond a certain Ct, the virus is no longer cultivable. This notion has already been known for a long time and is true for almost all viruses: the fewer viruses there are the more difficult it is to cultivate it ... However, I understand that this notion is important to promote this LAMP type system but I think you shouldn't be so exclusive and avoid extrapolating. Is there any literature linking Ct and infection in "real life" and not in vitro? This kind of study is obviously complicated to implement that's why your introduction and conclusion must be moderate. If a person in your family was at 30Ct, would you find it normal that she thinks herself negative with the behavior that this causes, moreover some patients may have been badly sampled. In short, you justified that your system is good at detecting contagious people despite your low sensitivity based on in vitro studies. I think it deserves to be qualified a little.

Finally here are some minor comments.

The CDC System reference is missing.

You should include earlier that the heating is used for inactivation of biological samples in the field.

Line 212 there is a problem with the text font size

Your percentages are oddly expressed. You should rather focus on the samples> 106 which you are supposed to detect (63/77, 83% 46/53, 87% 18/25, 72%)

Figure 4, how do you explain that samples at 25CT are not detected?

The paragraph on saliva for me is not of interest, if you think that the conditions of transport and storage are not comparable it is useless to develop this part further. You should simply delete it.

Reviewer #2: The manuscript entitled “Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab and saliva samples” by Dudley and colleagues describes the analytical sensitivity of previously published RT-LAMP assays for SARS-CoV-2. While the manuscript is well prepared, the study has little novelty and several major concerns:

Major concerns:

1. The author’s interpretation in relation to the poor sensitivity of the assays (1x10^5-1x10^6 copies/ml) they have evaluated is not fully justified. Although in vitro studies showed no or low rates of recovery of replication competent virus in cell culture from specimens with weaker RT-qPCR CT values or lower viral loads, the purpose of COVID-19 testing is not only to determine the transmissibility of the virus. The analytical sensitivity of COVID-19 tests is critical to identify pre-symptomatic cases, to identify patients with severe disease who may have cleared the virus from upper respiratory samples but is still sick from COVID-19 and also for epidemiological purposes. With such a poor sensitivity of their assays it is likely that a large number of tests will end up with false negative results with adverse outcomes.

2. The authors claim to apply their assay as POC is not very encouraging considering the fact that switching to colorimetric detection methods will further reduce the sensitivity of the test.

3. The specificity of any of the assays were not addressed. It is not clear what is the rate of false positive results from these assays.

4. No proper clinical validation was done. So, the clinical performance characteristics of the assays in comparison with standard RT-qPCR assays are not clear.

5. It is somewhat surprising that the LOD for pure transcripts (Fig 1A) is two log higher than extracted RNA from samples (Fig 2B).

6. Different primer sets were tested in a much smaller set of samples compared to Gene-N-A primers.

7. Colorimetric saliva results were not correctly presented. The results need to be blindly called and then compared against NP swab RT-qPCR results.

8. There are now hundreds of RT-qPCR and RT-LAMP methods published including many extraction free PCR with much better analytical and clinical sensitivity. The authors need to discuss how their tests and evaluations provide additional benefits over existing assays.

**********

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Reviewer #2: No

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PLoS One. 2020 Dec 31;15(12):e0244882. doi: 10.1371/journal.pone.0244882.r002

Author response to Decision Letter 0

16 Dec 2020

Response to Reviewers

PLOS ONE Editor comments

*Please note that all line numbers correspond to the line numbers in the unmarked manuscript.

1. Please ensure that your manuscript meets PLOS ONE's style requirements

The documents have been updated to conform to the style templates provided.

2. Please clarify if the biological samples used in your study were:

(1) from an established biobank (if so please provide the name and a link)

(2) specifically collected for this study or not

(3) whether the samples were collected through a medically prescribed test

(4) whether the samples were completely de-identified before researchers accessed the samples.

The Sample Collection section was updated to include the requested information (page 5, lines 93-98).

3. Please provide the catalog number for the:

a) coronavirus qRT-PCR assay used in this study and

b) 4 Warmstart LAMP reagents and the included fluorescent dye (New England Biolabs, NEB).

These catalogue numbers have been included in the RT-LAMP section of the Materials and Methods (page 5, line 108 and page 6, line 117).

4. Please provide the sequence of the in vitro transcribed SARS-CoV-2 N gene RNA provided by Nathan Grubaugh.

The sequence for the in vitro transcribed SARS-CoV-2 N gene RNA is now included as S2 Table.

We included “data not shown” only for samples that tested negative by our assay that were also negative by diagnostic testing because there is nothing to show on a graph. We now include these negative samples on our table of samples and now refer to that data in S3 Table where the negative nature of the results from these samples is shown (page 10 line 199).

Reviewer comments:

Reviewer #1:

1. It is obvious that the system presented (without conventional prior DNA extraction) lacks sensitivity unlike the gold standards. The authors from the introduction endeavor to cite all the literature which justifies that beyond a certain Ct, the virus is no longer cultivable. This notion has already been known for a long time and is true for almost all viruses: the fewer viruses there are the more difficult it is to cultivate it ... However, I understand that this notion is important to promote this LAMP type system but I think you shouldn't be so exclusive and avoid extrapolating. Is there any literature linking Ct and infection in "real life" and not in vitro? This kind of study is obviously complicated to implement that's why your introduction and conclusion must be moderate. If a person in your family was at 30Ct, would you find it normal that she thinks herself negative with the behavior that this causes, moreover some patients may have been badly sampled. In short, you justified that your system is good at detecting contagious people despite your low sensitivity based on in vitro studies. I think it deserves to be qualified a little.

The authors agree with the sentiment of this reviewer. In order to address these concerns, the authors wish to de-emphasize identifying contagious individuals. It remains unknown what viral load threshold is required for onward transmission of SARS-CoV-2. While we don’t think anyone disagrees that identifying people with the highest viral loads is likely identifying those who are most contagious, we would like to instead emphasize the utility of this method to implement frequent surveillance for SARS-CoV-2. This assay is not as sensitive as qRT-PCR, but it is faster, cheaper, and more accessible allowing access to asymptomatic individuals who could be frequently tested. qRT-PCR, which cannot keep up with the demand for testing of symptomatic individuals and return results quickly, remains the gold standard but is itself insufficient for frequent surveillance testing.

The sensitivity of LAMP as described in this manuscript rivals other quick tests, such as good antigen tests, that are also in limited supply. There is growing evidence that less sensitive tests that can be administered multiple times per week may be more beneficial than a very sensitive test that provides results more slowly and is administered inconsistently.

The authors have updated the manuscript (changes to the abstract on page 2 lines 27-29 and 32-33, a paragraph removed in the introduction (page 3, paragraph between lines 49-50) and a new paragraph added (page 4, lines 69-81) into the introduction) to emphasize the value of a test like this to supplement diagnostic testing.

2. The CDC System reference is missing.

We have included the catalogue number for the components associated with the qRT-PCR assay that was used to generate viral loads in this manuscript (IDT, 10006770) (page 5, line 108). The protocol follows that of the CDC and we have included the reference to that protocol as well on page 5, line 108.

3. You should include earlier that the heating is used for inactivation of biological samples in the field.

Since heating was only used for the saliva-based system and it was suggested that this be removed from the manuscript, all references to heating the samples were removed.

4. Line 212 there is a problem with the text font size

The font size was fixed at old line 212 (new line 217).

5. Your percentages are oddly expressed. You should rather focus on the samples> 106 which you are supposed to detect (63/77, 83% 46/53, 87% 18/25, 72%)

We thank the reviewers for this suggestion, we agree that this would be helpful to emphasize throughout our comparisons. We did identify an error in that there were 78 samples with viral loads above the 1x10^3 copies/�l threshold from the primary samples in Figure 1B. We apologize for the error and have fixed this in the manuscript. To address this comment, we added a description about why we will focus on samples with viral loads greater than our threshold of 1x10^3 copies/ul to be more conservative than the transcript estimate of 625 copies/ul on pages 9-10, lines 193-195. We then incorporated the percentages of samples that were positive above our 1x10^3 copies/ul limit of detection for each condition we tested and highlighted the improvements these conditions made to the detection of samples that fell at or above this limit of detection. This can be found in lines 224-226, 264-266, and 360-361.

6. Figure 4, how do you explain that samples at 25CT are not detected?

The ability to detect primary swab samples that have not been purified, but are added directly into the RT-LAMP reaction, is dependent not only on the concentration of the virus in the sample, but also the inhibitors in the sample. Figure 1 shows that there are many undetectable samples with viral loads above the level we believe we can detect with purified RNA. This is also very evident in Figure 2B. When we isolate RNA and purify it away from inhibitors of the primary sample, we detect everything above the 156 copies/ul threshold.

The variability in nucleases and the media the samples were collected in in the primary sample means that different thresholds of virus may be detectable in different samples. Specifically related to Figure 4, this is the only dataset we were unable to acquire in-house viral loads representative of the viral load of the sample at the time of our assay. Storage for days at 4C and a freeze/thaw of the samples occurred between the time the Ct value was acquired at the hospital and the time that we ran the samples in this assay. Therefore, several samples were equivocal at a Ct of ~25 (all samples were positive in at least 1 replicate), which is likely due to a combination of inhibitors in the samples and the handling of these particular samples between the assay providing the Ct value and our assay. For all other datasets in the manuscript we obtained in-house viral loads after the same storage conditions as our RT-LAMP assay to compare our data for quantitative purposes. This caveat about the data presented in Figure 4 has been included in the text on pages 15 and 16, lines 322-327 and further discussion of the role of inhibitors are included on page 17, lines 352-357.

7. The paragraph on saliva for me is not of interest, if you think that the conditions of transport and storage are not comparable it is useless to develop this part further. You should simply delete it.

The authors have removed the reference to the saliva-based version of this test throughout the manuscript and will describe this in greater detail elsewhere (1).

Reviewer #2:

We thank the reviewer for this perspective. We completely agree that a diagnostic test is ideally sensitive enough to detect individuals with low viral loads. But even now, months after our initial submission, diagnostic qRT-PCR tests are still not widely available enough and the test presented in this manuscript can help fill in specific gaps in testing.

RT-LAMP represents a low-cost, faster turn-around time, lower sensitivity test that can be used to screen individuals frequently who are otherwise asymptomatic and do not qualify for diagnostic testing at many sites that continue to have to limit the number of daily tests they run (2). Asymptomatic individuals have similar viral loads as symptomatic individuals and would be just as likely to test positive by this assay as symptomatic individuals. The use of the newly FDA emergency use-approved antigen tests, as another version of a lower sensitivity, fast turn-around test, has been promoted by the CDC as a way to augment testing (3). RT-LAMP has a similar capability to detect the individuals with high viral load who are likely the most contagious individuals. We have changed the emphasis of the importance of this test away from identifying highly contagious individuals and incorporated the importance of frequent screening and how this test can help in that capacity. This will be especially important as the massive fall/winter surge in the US declines after the arrival of vaccines and vigilance will be required to identify and suppress outbreaks from asymptomatic individuals throughout society.

These changes are presented in the introduction in lines 69-81. In addition, we have transitioned our figures and analysis to report the sensitivity of this assay as copies/ul rather than copies/ml. This transition is in line with most other RT-LAMP-based manuscripts and the way that the CDC reports the limit of detection of their PCR-based assay. This will hopefully avoid confusion when comparing these data with those of other published work. The sensitivity of our assay is not different from other RT-LAMP-based assays that do not use an RNA extraction first (~100 copies/ul).

2. The authors claim to apply their assay as POC is not very encouraging considering the fact that switching to colorimetric detection methods will further reduce the sensitivity of the test.

Based on the recommendation of the reviewers, the authors are removing the colorimetric test results with saliva from this manuscript as it is outside of the scope of this work. To be clear, creating a test that can be simple enough to run and does not require expensive equipment, such as a lightcycler, are important steps to getting widespread SARS-CoV-2 screening available to schools and workplaces, who can run these tests on-site with some training. If we can screen workforces or schools and catch at least some of the most highly positive individuals, we hypothesize this will help reduce transmission risk, though we cannot predict by how much. More sensitive options are by nature too costly and time consuming to use in this capacity. This allows us to cast a much wider net with and assay that is comparable in sensitivity to antigen tests approved by the FDA, which also remain in limited supply and are not widely available. The advantages of this test, even if less sensitivity than qRT-PCR, are addressed in lines 69-81.

3. The specificity of any of the assays were not addressed. It is not clear what is the rate of false positive results from these assays.

The specificity of the test is determined by the primer sets used in the assay. Because the primer sets tested in this manuscript were previously published and many were shown to be specific for SARS-CoV-2 either in silico by sequence analysis or by testing against other coronaviruses in the lab, we did not reevaluate this. We have added a paragraph to the methods section about the specificity of the primer sets based on previous work by those who developed the primers in lines 137-146. We also added citations for each primer set in Table S1 for additional reference. We did test 5 samples from the hospital that were negative for SARS-CoV-2 by diagnostic tests but were positive for other respiratory pathogens that all tested negative by our LAMP assay using the gene-N-A primers. We included more details about these samples in lines 200-203. In addition, we tested 26 other SARS-CoV-2 negative samples from the hospital with the gene-N-A primers and all samples also tested negative by our LAMP test. Based on these results, specificity of the assay with the gene-N-A primers is 100% for SARS-CoV-2. We have added this to the text in lines 202-203. Unfortunately, we do not have access to a large number of other coronaviruses and primer sets to adequately address specificity. The primers described by Color genomics, which we recommend using based on the experimental results provided in our manuscript, were validated against 51 organisms and showed those primers were highly specific for SARS-CoV-2 as part of the EUA process for their assay that also uses these primers. This information was added in lines 145-146.

4. No proper clinical validation was done. So, the clinical performance characteristics of the assays in comparison with standard RT-qPCR assays are not clear.

The authors acknowledge that we did not have the samples nor resources to provide a clinical validation of this particular assay. The authors are recommending this test as a non-diagnostic surveillance test, which does not require strict clinical validation. Instead, the authors wished to present their findings to provide their insight to the community at large who may be interested in developing this test in any number of capacities. If someone wishes to pursue this test in a clinical or diagnostic capacity, additional work would be required. This has been added to the discussion in lines 406-407.

5. It is somewhat surprising that the LOD for pure transcripts (Fig 1A) is two log higher than extracted RNA from samples (Fig 2B).

Thanks for pointing out this inconsistency. By pure transcript we can detect as low as 156 copies/ul (Figure 1) and with extracted RNA we can detect samples with a concentration as low as 10 copies/ul and consistently detect samples above 23 copies/ul. Please note that the RNA copies/ul reported is the concentration of the starting sample in Fig. 2B. During the process of RNA isolation, the RNA is purified and concentrated by 3-fold and then 5ul were used to emulate the volume and copies of RNA that goes into qRT-PCR for comparison. For a sample with 10 copies/ul, the total copies per reaction would be 10 copies RNA x 3-fold dilution x 5ul=150 copies/rxn. That is essentially the same number of copies/reaction detectable with the transcript where 1ul of a sample with 156 copies of RNA/ul were added. Overall, detection in transcript mimics that of detection in primary samples where RNA was isolated. This distinction between copies of RNA/ul in the starting sample and copies of RNA/reaction is important when interpreting the data shown in Figure 2B and for clarity we’ve added this description to the text in lines 257-264.

6. Different primer sets were tested in a much smaller set of samples compared to Gene-N-A primers.

It is true that we focused on the Gene-N-A primers for much of their work, including the work with primary samples. Due to limitations in acquiring primary samples and that they were used for multiple assays and were used up, we were not able to repeat some of our initial work with the alternative primers that we believe perform better than the Gene-N-A primers. Instead, we chose to share the work we did to determine whether different primer sets could improve the efficiency of the assay using standards and samples that we did have available.

7. Colorimetric saliva results were not correctly presented. The results need to be blindly called and then compared against NP swab RT-qPCR results.

Per request from the reviewers the colorimetric saliva results were removed from the manuscript.

This is a rapidly changing field and while at this point, there are other extraction-free PCR test options, limits of detection around 100 copies/ul are common with most of these methods. The primary contribution of this manuscript is that it shows results from performing the assay on primary NP swab samples. Most other publications utilize contrived samples with either virus or transcript that do not necessarily recapitulate the performance of the assay in primary samples containing biological inhibitors such as nucleases that will alter the performance (addressed in lines 64-68 and 341-348). While our LOD with transcript is around 156 copies/ul, in primary samples that falls closer to 1,000 copies/ul.

As more assays evolve it is likely that modifications will improve this LOD and the authors believe it is important to present what we’ve learned to the community in order to aid in that development as quickly as possible. Lastly, as mentioned in response to question 1, the benefit of an assay like this that does not require extraction first is that is faster and cheaper than more sensitive molecular tests, which means it can be performed much more frequently and is likely to capture those that are most likely to transmit the virus despite being asymptomatic who are otherwise not getting tested. Different versions of assays may all have utility, as end-user preferences will likely factor into eventual acceptance for repeated testing. This emphasis has been incorporated into the introduction (lines 69-81).

Footnotes:

1. https://www.medrxiv.org/content/10.1101/2020.07.28.20164038v2.

2.https://www.pewtrusts.org/en/research-and-analysis/blogs/stateline/2020/08/14/to-speed-up-results-states-limit-covid-19-testing; https://www.businessinsider.com/why-states-like-new-york-are-limiting-covid-19-tests-2020-3; https://www.wsj.com/articles/covid-19-testing-is-hampered-by-shortages-of-critical-ingredient-11600772400

3. https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antigen-tests-guidelines.html

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PLoS One. doi: 10.1371/journal.pone.0244882.r003

Decision Letter 1

Ruslan Kalendar

18 Dec 2020

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples

PONE-D-20-29372R1

Dear Dr. Dudley,

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PLoS One. doi: 10.1371/journal.pone.0244882.r004

Acceptance letter

Ruslan Kalendar

23 Dec 2020

PONE-D-20-29372R1

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples

Dear Dr. Dudley:

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.

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Associated Data

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

Supplementary Materials

S1 Fig. Comparison of Gene-N-A, Color-N, and Color-E primers to several ORF1a -targeting primer sets and combinations with two primary samples and irradiated SARS-CoV-2.

Samples that were not detectable were plotted on the ND line set at Cq 80, the hißghest cycle number in our assay.

(TIF)

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S1 Table. Primer sequences and final 1X concentrations used in the RT-LAMP reactions.

(DOCX)

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S2 Table. Nucleotide sequence of the RNA transcript provided by Dr. Nathan Grubaugh.

(DOCX)

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(DOCX)

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Data Availability Statement

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PERMALINK

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples

Dawn M Dudley

Christina M Newman

Andrea M Weiler

Mitchell D Ramuta

Cecilia G Shortreed

Anna S Heffron

Molly A Accola

William M Rehrauer

Thomas C Friedrich

David H O’Connor

Roles

Abstract

Introduction

Materials and methods

Sample collection

qRT-PCR

RT-LAMP

Sample lysis

Statistical analysis

Results

Limit of detection with RNA transcript and Gene-N-A primers

Fig 1. Detection of SARS-CoV-2 by RT-LAMP from transcript or primary NP swab samples.

Limit of detection with primary nasopharyngeal swab samples

RT-LAMP is inhibited by adding larger volumes of primary sample

Lysis buffer improves the sensitivity of RT-LAMP

Fig 2. Comparison of the detection of SARS-CoV-2 positive primary NP swab samples after using direct sample addition to either LucigenQE treatment or isolated vRNA.

RNA isolation improves the limit of detection of RT-LAMP to levels similar to qRT-PCR

Alternative primers improve efficiency

Fig 3. Comparison of RT-LAMP Cq value on primary NP swab samples, irradiated SARS-CoV-2 or SARS-CoV-2 TWIST RNA amplified with different primer sets.

Fig 4. Comparison of the best-performing primers and combinations on additional primary NP swab samples with varying levels of virus with and without GuHCl.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Ruslan Kalendar

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Author response to Decision Letter 0

Decision Letter 1

Ruslan Kalendar

Roles

Acceptance letter

Ruslan Kalendar

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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