key: cord-0947818-wr4jhjfy
authors: Zhao, W.; Tang, G.; Zhang, Z.; Tan, W.; Fei, L.; Sun, J.; Li, Y.; Zou, S.; Yang, Y.; Cai, K.; Li, S.; Wang, Z.; Liu, J.; Mao, G.; Ma, Y.; Zhao, G.-P.; Tian, Z.-G.
title: RT-RPA-Cas12a-based discrimination of SARS-CoV-2 variants of concern
date: 2022-05-16
journal: nan
DOI: 10.1101/2022.05.11.22274884
sha: bbb040d8fa5e5c9a3a275773bbe03a129e7e58ee
doc_id: 947818
cord_uid: wr4jhjfy

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. However, current methods are limited by the low sensitivity, long turn-around time or high cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 VOCs by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 204 clinical samples, showing 99% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and {Delta}H69-V70 were discriminated by RRCd, demonstrating 100% accuracy in clinical samples with Ct <33. The method completes within 65 min and could offer visible results without using any electrical devices, which may facilitate point-of-care testing of SARS-CoV-2 and its variants.

The detection limit of RRCd 143 To determine the limit of detection (LoD) of RRCd, we built a standard curve to 144 dissect the correspondences between the viral copy numbers and the Ct values using 186 in both readouts (Ct < 33) ( Table 2) . 187 We next evaluated the performance of RRCd on discrimination of SARS-CoV-2 Figure 5A, Supplementary Figure 10) . However, the discrimination ability 195 decreased strikingly when testing the samples with Ct values beyond 33, which is 196 possibly due to the relatively high LoD of S gene 501 as mentioned above (Figure 3) . 197 The 478 position of S gene could be used as a target to identify Delta and 198 Omicron VOCs (Figure 5B, Supplementary Figure 11) (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 12) . Although a relatively small sample 227 size was used, these results indicated that the whole process of RRCd including the 228 RT-RPA amplification and the lateral-flow detection is independent of electrical 229 devices, which may facilitate the development of POCT products (Table 1) . 230 In addition, the RRCd assay has the potential to be easily adapted to detect (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. showed that these pairs of RT-RPA primers hit 99.7% of the genome sequences, 287 demonstrating a good applicability in detection of prevailing clinical samples. 288 Optimal crRNAs for the discrimination of S gene 501 variants were assessed 289 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Then, the crRNAs were purified using RNA Clean and Concentrator kit (Cat # R1017, 307 Zymo Research). The concentration of crRNA was quantified by NanoDrop 2000C 308 spectrophotometer (Thermo Scientific) and stored at -80°C before use. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274884 doi: medRxiv preprint and then cloned into a T7 promoter expression plasmid to be used as the templates for 364 IVT of viral RNAs, respectively. The transcribed RNAs were detected using 365 crRNA-SARS-CoV2 N by the RRCd assay as described above. lateral-flow readouts were analyzed as described above. For the dilutions (1 × 10 7 , 1 384 × 10 6 , 1 × 10 5 , 1 × 10 4 , 1 × 10 3 and 1 × 10 2 copies per reaction), at least three 385 independent experiments were performed, while for the dilutions with 10 and 5 386 copies per reaction, at least five independent experiments were conducted. 387 RNase-free water was used as the negative control. 388 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The corresponding RT products were used as inputs to the RPA assay and 421 subsequent products were analyzed by fluorescence based CRISPR assay as (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The RT-RPA products were analyzed by fluorescence based CRISPR assay as LoD, limit of detection (copies per reaction); NA, not applicable. 670 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; Table 2 Total PPA/NPA 88% 100%

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274884 doi: medRxiv preprint motif; VOC, variant of concern; WT, wild type; GNP, gold nanoparticle; gt, goat. 690 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274884 doi: medRxiv preprint S gene T478 NC 5 10 7 10 4 10 3 10 2 10 10 6 10 5 NC 5 10 7 10 4 10 3 10 2 10 10 6 10 5 NC 5 10 7 10 4 10 3 10 2 10 10 6 10 5 NC 5 10 7 10 4 10 3 10 2 10 10 6 10 5 NC 5 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274884 doi: medRxiv preprint

Recurrent emergence of SARS-CoV-2 spike 600 deletion H69/V70 and its role in the Alpha variant B.1.1.7

The next phase of SARS-CoV-2 surveillance: real-time molecular 604 epidemiology

Comparative 609 analysis of the risks of hospitalisation and death associated with SARS-CoV-2 omicron 610 (B.1.1.529) and delta (B.1.617.2) variants in England: a cohort study

The next phase of SARS-CoV-2 surveillance: real-time 614 molecular epidemiology

Clinical validation of a Cas13-based 623 assay for the detection of SARS-CoV-2 RNA

COVID-19: Rapid antigen detection for 626 SARS-CoV-2 by lateral flow assay: A national systematic evaluation of sensitivity and 627 specificity for mass-testing

DNA detection using 630 recombination proteins

Detection of a SARS-CoV-2 variant of concern in South Africa

CDetection: 648 CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base 649 specificity

Increased yield of 651 PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis 652 system

detection of SARS-CoV-2 S gene 501, using fluorescence-based readouts. The 697 dsDNA derived from pseudovirus S gene N501 or Y501 was incubated with Cas12a, 698 FQ-ssDNA, and distinct crRNA at 37°C, and the corresponding fluorescence signal 699 was recorded over 60 min (left panel) and quantified at 30 min (right panel)

The samples analyzed were 703 the same as those in panel B. The reactions were conducted at 37°C for 30 min before 704 the mixtures were transferred onto the strips, using FB-ssDNA probe instead of 705 FQ-ssDNA. The relative greyness (G) of strip bands was quantified using ImageJ. In 706 this study, GSample / GNC of the T band ≥ 1.5 indicates positive. D, E. Specificity 707 analysis of RRCd in detection of SARS-CoV-2 S gene 478. F, G. Specificity analysis 708 of RRCd in detection of SARS-CoV-2 S gene 69-70. H. Schematic of RRCd in 709 detection of SARS-CoV-2 VOCs by Cas12a cis-cleavage activity. I, J. The specificity 710 was demonstrated by PAGE analyses

No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. respectively. The relative fluorescence intensity (F/F0) was calculated as the 719 fluorescence signal versus the starting signal (left panel). Correspondingly, the 720 fluorescence signal at the 30 min-reaction was quantified (middle panel). The positive 721 samples of lateral-flow readouts (right panel) are indicated as blue by the T-line 722 quantification as described. Data are presented as mean ± S.D. from at least three 723 independent experiments. For the dilutions with 10 and 5 copies per reaction, at least 724 five independent experiments were conducted. A. LoD determination of RRCd for E 725 gene

No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. discriminated by crRNA-N501 (green) and crRNA-Y501 (light blue) using 753 fluorescence-based readouts

The analysis results are shown as the 756 number of false discriminants and the false-discriminant rate for each Ct range on the 757 bottom of the panel. B. Discrimination of SARS-CoV-2 S gene 478 variants in 758 clinical samples. In total, 21 positive samples (Ct range of 17-34) and 19 negative 759 samples were detected. The S gene 478 variants were discriminated by crRNA-T478 760 (blue) and crRNA-K478 (pink) by fluorescence readouts. The lateral-flow readouts 761 using crRNA_T478 (left) and crRNA_K478

The corresponding SARS-CoV-2 763 pseudoviruses were used as the positive controls. The full datasets for panel a and b 764 are shown in Supplementary Figure 10 and Supplementary Figure 11

No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity