key: cord-0738516-3qi7dh8n
authors: Brandsma, Eelke; Verhagen, Han J M P; van de Laar, Thijs J W; Claas, Eric C J; Cornelissen, Marion; van den Akker, Emile
title: Rapid, sensitive and specific SARS coronavirus-2 detection: a multi-center comparison between standard qRT-PCR and CRISPR based DETECTR
date: 2020-10-10
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa641
sha: 38156d59afffd017c256f00202da1ca5fcaf9b61
doc_id: 738516
cord_uid: 3qi7dh8n

Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of isothermal reverse transcriptase loop mediated amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to qRT-PCR without sacrificing sensitivity/specificity. Here we compare DETECTR with qRT-PCR to diagnose COVID-19 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared to qRT-PCR, however, this was not confirmed in this large patient cohort, were we report 95% reproducibility between the two tests. These data showed that both techniques are equally sensitive in detecting SARS-CoV-2 providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different gRNAs can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point of care test (POCT), showed a 100% correlation to the high-throughput DETECTR assay. Importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses. As there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR-platforms for routine non-SARS-CoV-2 diagnostic testing.

SARS Coronavirus-2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), emerged in December 2019 in Wuhan, China and caused a pandemic. As of July 19th 2020, over 14 million confirmed SARS-CoV-2 infections and more than 600.000 COVID-19 related deaths have been reported worldwide. To curb this epidemic, effective prevention and control measures including the early identification of SARS-CoV-2 infected individuals, are crucial. Outbreak management is hampered by the high transmissibility and broad spectrum of clinical features of SARS-CoV-2. Severe illness marked by pneumonia, acute respiratory distress syndrome (ARDS) and the need for mechanical ventilation is strongly skewed towards people over 70 years old and those with underlying diseases. Many others experience only mild to moderate symptoms such as fever, fatigue, (dry) cough and/or dyspnoea or do not have complaints at all [1] .

Infection surveillance and notification play an important role in outbreak prevention and control. As many infections may go unnoticed, large-scale availability of reliable diagnostic tests also for those with mild symptoms is of critical importance to protect especially those at highest risk of developing severe illness. Accurate monitoring of the SARS-SoV-2 epidemic curve helps estimating future disease burden and serves as an important societal impact parameter for pre-emptive policy making e.g. with regards to the justification of less or more restrictive quarantine measures and prevention of health-care system overflow [2] [3] [4] . Reverse transcriptase polymerase chain reaction (RT-PCR) is the current diagnostic standard for the detection of SARS-CoV-2. Despite its high sensitivity and specificity, qRT-PCR requires (expensive) specialized equipment, trained staff, and has a relative long turn-around-time (TAT; 2-4 hours). In the Netherlands, the strong dependence on qRT-PCR caused a shortage of reagents and consumables during the pandemic, which limited the test-capacity and resulted in possibly suboptimal outbreak management.

Isothermal reverse transcriptase loop mediated isothermal amplification (RT-LAMP) in combination with Cas12 detection does not need expensive specialized equipment, is highly sensitive and specific, A c c e p t e d M a n u s c r i p t 4 has a short TAT and is easy to implement and therefore could be used as an alternative for qRT-PCR [5, 6] . This technology is termed DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR). The single strand DNA nuclease activity of Cas12 can generate a high-throughput SARS-CoV-2 point-ofcare test (POCT) without aspecific amplification as observed with RT-LAMP using intercalating fluorescent dyes or turbidity readouts [5, 7] , review see [8, 9] . Since DETECTR depends on both signal amplification by RT-LAMP and reporter degradation after Cas12-dependent amplicon recognition ( Figure 1) , the assay produces a binary readout and is potentially more sensitive and specific compared to qRT-PCR [5, 6] . A direct comparison between qRT-PCR and this novel DETECTR assay on a large patient cohort has not yet been performed. In the Netherlands, patients suspected of COVID-19 are admitted under strict isolation procedures to prevent nosocomial transmission of SARS-CoV-2 within the hospital. Unnecessary isolation measures pose a significant burden on the nursing staff as well as on the capacity and costs of the hospital. A rapid highly sensitive SARS-CoV-2 assay, preferably suitable as a POCT, would be of added value for (rapid) clinical decision-making and the optimization of patient flow within the hospital. In this manuscript we describe the development of an in-house SARS-CoV-2 DETECTR assay, compare its performance with routine diagnostic qRT-PCR on almost 400 patient samples of three Dutch hospitals, thereby providing a first field test of this novel Cas12-mediated SARS-CoV-2 detection tool.

All specific information on reagents and relevant concentrations are listed in supplemental tables

Primers (supplementary Table 1 .1) were dissolved in ultrapure water to a final concentration of 100 µM and prepared in 10x primer master mixes (supplementary Table S1 .2). For isothermal amplification, 15 µl of complete RT lamp reaction mix was prepared on ice (supplementary Table   S1 .3) and incubated with 10 µl of isolated RNA or DNA CTRL plasmid at 62 o C.

A c c e p t e d M a n u s c r i p t 5

RNA extracts derived from COVID-19 positive patients were run in a reverse transcriptase (RT) reaction according to table 1.6 and hence amplified with or without PCR. Next, qRT-PCR as well as RT products were incubated with N-gene RNPs and analyzed via HT-detection as described below.

RNPs were formed by incubating LbCas12 (supplementary Table S1 .4) with targeting Guide RNAs in a RNP reaction mix for 30 min at 37 o C (supplementary Table S1 .5) and subsequently, probe 1,2 or 3 was added in a final concentration of 100 nM (probe 1 and 3) or 500 nM (probe 2).

2,5 µl of RT-LAMP reaction mix was incubated with 22,5 µl of RNP complex containing probe 1 or 3, at 37 o C for 10 minutes in chimney multi-well plates covered with seals. Readout was performed after 10 minutes of incubation, unless indicated differently in the figure legends, at 37 o C in a Biotek Synergy 2 plate reader using a 485/20 excitation and a 528/20 emission filter. lateral chip assay 2 µl of RT-LAMP reaction mix was incubated with 20 µl of RNP complex containing probe 2, at 37 o C for 10 minutes. Next, 80 µl NEBuffer2.1 (1x concentrated) was added. Lateral flow strips were incubated for 2 minutes at RT allowing liquid to migrate. Readout was performed visually.

All data was first tested for normality by the Shapiro Wilk test (p=0,05). Data with a gaussian distribution was analyzed with an unpaired two-sided student's t-test in case of the comparison of 2 samples or an one-way ANOVA with a Dunnett's post-test in case of 3 samples or more. Data which did not follow a gaussian distribution and contained 3 groups or more, was analyzed with a Kruskall-A c c e p t e d M a n u s c r i p t 6 Wallis test followed by a Dunnett's post-test. All statistics were analyzed in Graphpad Prism version 8.0.2.

The majority of patient samples were nasopharyngeal swabs in transport medium, the remainder were either broncheo-alvealar lavage (BAL) or sputum. Extensive description of RNA isolation and qPCR methods can be found in supplemental methods.

Both (RT-)LAMP and Cas12-RNPs can be used to detect RNA/DNA, while the combination potentially increases sensitivity and specificity [6] . We compared the sensitivity of RT-LAMP, RT-Cas12-RNP and DETECTR (combination RT-LAMP/RT-Cas12-RNP, Figure A c c e p t e d M a n u s c r i p t 7 nucleotides (nt). The use of a 12 nt probe increased the signal to noise ratio but not the sensitivity of the test (Supplemental Figure 2A -B). The plateau of the fluorescent signal using DETECTR is reached after 10 minutes. However, >75% of the maximum fluorescence is reached within 5 minutes, suggesting that the assay can be performed faster if required (Supplemental Figure 2C ). Longer incubation does not increase the fluorescent signal (Supplemental Figure 2D ). However, plates can be re-measured or stored for at least three days without significant loss of signal when stored at room temperature in ambient light (Supplemental Figure 2D ). In conclusion, our DETECTR data confirm short turn-around-times (<30 minutes including RT-LAMP), signal robustness and ease of result interpretation. (potential) inhibitory factors present in patient material have also been diluted. As the Cas12-RNP complex is single nucleotide sensitive [11, 12] , mutations within the gRNA recognition site may prevent Cas12 detection. Using a dual target approach with gRNAs that anneal to distinct parts of the RT-LAMP generated amplicon could prevent escape from Cas12 detection (supplemental figure   1B ). DETECTR results with gRNA1, gRNA2 and combined gRNA1/gRNA2 yielded similar results ( Figure   4B ) Most DETECTR results were obtained using a high throughput 96/384 wells spectrophotometer to detect the cleaved fluorescent probe. A major advantage of DETECTR is that it can be used as an individual POCT using lateral flow strips for read out. Individual lateral flow results (n=40) were 100% concordant with the high throughput results (supplemental figure 4) . To confirm robust signals in 'difficult' clinical samples, we analyzed 8 samples with not interpretable qRT-PCR results using spectrophotometric and lateral flow detection. Again, fully concordant results: SARS-Cov-2 positive (n=4) and SARS-CoV-2 negative (n=4) (Supplemental Figure 4C) . The binary readout is easy to interpret, irrespective of readout method or Cq-value. Therefore, DETECTR POC tests could be used in low-resource countries/regions or as a fast and reliable equipment independent confirmation test to confirm ambiguous qRT-PCR samples.

In summary, here we compared DETECTR with qRT-PCR for SARS-CoV-2 diagnosis in a large patient cohort over multiple hospitals and report a 95% accordance. These data are in line with recently published studies where cohorts were tested in a single institute [5, 13] .These data are in line with a recently published study where only a small cohort (83 samples) was tested derived from a single hospital In addition, our data suggest that a 12nt probe is superior over a 8nt probe and we suggest to use a double guide approach to prevent escape from DETECTR due to mutations within amplicons. Overall, DETECTR has comparable sensitivity and superior specificity to qRT-PCR. Our results show that DETECTR represents a reliable, cheap, fast and technically independent alternative to complement qRT-PCR platforms. The low-demand on facility equipment, especially concerning the POCT, makes DETECTR especially suitable for resource low countries/regions. In this paper we A c c e p t e d M a n u s c r i p t 10 show a LOD for RT-LAMP at 500 copies and for DETECTR at 50 copies. It is however important to note that we have defined the LOD on N-gene plasmids instead of synthetic SARS-CoV-2 RNA. This makes the comparison between RT-LAMP and DETECTR independent of reverse transcriptase efficiency. However, it may not accurately display the LOD of RNA samples, since efficiency to convert RNA to DNA by reverse transcriptase also depends on secondary RNA-structure and sample matrix. Other studies have however shown a similar LOD for RT-LAMP as reporter here after spiking synthetic viral RNA into different matrices, such as mucin or blood [10] . A current limitation of DETECTR is the dependence on three separate reactions, namely RNA isolation, RT-LAMP amplicon amplification and Cas12 mediated reporter degradation. The latter has to be considered a step back in comparison to qRT-PCR, where post amplification handling, a major risk in causing false positive results by contamination, could be removed from the workflow. Further research should focus on integrating all DETECTR steps, including RNA-isolation, into the same reaction tube without post amplification processing. In the current study, the extracted RNA used as input for qRT-PCR was also used for DETECTR. Excitingly, in a recent paper published during the review process, the use of a heat stable Cas12 from Alicyclobacillus acidiphilus potentially makes combining the RT-LAMP and Cas12 reactions in one tube possible, which was verified in a test panel including 200 positive patient samples [13] . In addition, these authors showed that RT-LAMP multiplexing of various internal control amplicons together with the viral amplicon in one reaction may be possible, further adding to the robustness of assay results. Of note, onestep RT-LAMP approaches including various RNA extractions have been developed, e.g. for Zika virus [14, 15] , and compatibility with DETECTR will need to be determined. However, Joung et al. showed that RNA isolation may need to be carried out separately from the RT-LAMP and Cas12 reactions to maintain optimal sensitivity [13] . Importantly, as detection is not compromised upon diluting patient material 10-100 times, the technique may allow the implementation of pooled sample approaches in low-prevalence regions/countries significantly increasing testing capacity (e.g. 20 samples without loss of detection). However, it must be noted that in this patient cohort DETECTR and qRT-PCR were performing on parity. The clinical A c c e p t e d M a n u s c r i p t 11 sensitivity of DETECTR could be lower despite its higher analytical sensitivity ( Figure 1F, 1H) due to the matrix of clinical samples having a more profound inhibitory effect on DETECTR technology. Importantly, once implemented the suggested approach can be easily diverted to screen other existing or emerging pathogens or any other platform that requires identification based on specific DNA/RNA [6, 11, 12] . The DETECTR test helps to optimize diagnostic strategies for both bedside and high-throughput settings leading to an increase in testing capacity and improved diagnostic evaluation, ultimately leading to better determination of endemic progression facilitating governmental policy decisions.

A c c e p t e d M a n u s c r i p t 12

This study was supported by a NWO-ZONMW "creative solutions to battle COVID-19" grant from the Dutch government (No.5000.9954). We would like to thank the individuals part of the studied cohort.

EB and HV performed the RT-LAMP, DETECTR assays. MC, TvdL, EC and JS collected the cohort material, isolated RNA and performed the qRT-PCR on validated platforms. EvdA supervised the study. All authors contributed in writing the manuscript, which was critically reviewed by all authors.

The authors report no conflict of interest confirmed by qRT-PCR were found to be negative using SARS-CoV-2 specific DETECR. ****=p<0.0001 using two-sided unpaired T-test.

A) 378 qRT-PCR confirmed SARS-CoV-2 tested samples from different centers were compared to the results obtained using DETECTR. The matrix displays the results from the DETECTR assay (vertical) A c c e p t e d M a n u s c r i p t 18 

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