Rapid screening for SARS-CoV-2 variants of concern in clinical and environmental samples using nested RT-PCR assays targeting key mutations of the spike protein

Abstract

New SARS-CoV-2 mutations are constantly emerging, raising concerns of increased transmissibility, virulence or escape from host immune response. We describe a nested RT-PCR assay (~1500 bps) to detect multiple nucleotide changes resulting in key spike protein mutations distinctive of the major known circulating SARS-CoV-2 variants, including the three Variants of Concern (VOCs) 20I/501Y.V1 (United Kingdom), 20H/501Y.V2 (South Africa), and 20 J/501Y.V3 (Brazil), as well as the 20E.EU1 variant (Spain), the CAL.20C recently identified in California, and the mink-associated variant (GR, lineage B.1.1.298). Prior to application to field samples, the discriminatory potential of this PCR assay was explored using GISAID and Nextclade. To extend variant detection to challenging matrices such as sewage, where the amplification of long fragments is problematic, two short nested RT-PCR assays (~300 bps) were also designed, targeting portions of the region spanned by the long nested assay. The three newly-designed assays were then tested on field samples, including 31 clinical samples (7 fully-sequenced swab samples, and 24 uncharacterized ones) and 34 urban wastewater samples, some of which collected in areas where circulation of VOCs had been reported. The long assay successfully amplified 29 of the 31 swabs (93%), allowing the correct identification of variants 20I/501Y.V1 and 20E.EU1 present in the panel of previously characterized samples. The Spanish variant was detected in 14/24 of the uncharacterized samples as well. The sequences obtained using the short assays were consistent with those obtained with the long assay. Mutations characteristic of VOCs (UK and Brazilian variant) and of other variant (Spanish) were detected in sewage samples. To our knowledge, this is the first evidence of the presence of sequences harboring key mutations of 20I/501Y.V1 and 20 J/501Y.V3 in urban wastewaters, highlighting the potential contribution of wastewater surveillance to explore SARS-CoV-2 diversity. The developed nested RT-PCR assays can be used as an initial rapid screening test to select clinical samples containing mutations of interest. This can speed up diagnosis and optimize resources since it allows full genome sequencing to be done only on clinically relevant specimens. The assays can be also employed for a rapid and cost-effective detection of VOCs or other variants in sewage for the purposes of wastewater-based epidemiology. The approach proposed here can be used to better understand SARS-CoV-2 variant diversity, geographic distribution and impact worldwide.

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…Specificity: Unclear [309] Almost all variants 69del, 70del, Y144del, N501Y and A570D Compared to WGS, RT-PCR and sanger sequencing require shorter time and lower cost. LOD unclear [310] SNP genotyping Almost all variants Unreported >62% to be able to distinguish two variants of different genotypes [311] RT-LAMP P.1 (Gamma), P.2 (Zeta) N501Y, E484K/Q, K417N/T Sensitivity: 97%, Specificity: 100% (based on N/E targets) [312] B.1.1.7 (Alpha) 69del, 70del LOD: 39-10,000 RNA copies/reaction [313] CRISRP/Cas-based technique B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) N501Y LOD, sensitivity and specificity unclear [346] Almost all variants have D614G D614G LOD: 10 RNA copies/reaction [314] Other variants E174R/S542R/K548R, S254F LOD: 50-1000 RNA copies/reaction, specificity: 100% [315] Almost all variants have D614G D614G LOD: 82 RNA copies/reaction, % specificity unclear [155] Antigen test B.1.1.7 (Alpha), B.1.351 (Beta), Unclear LOD: 1.7×10 5 – 6.6×10 7 RNA copies/mL [316] Sensitivity and specificity: Unclear Antigen and antibody test B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) Unclear Detection rate of antigen/antibody tests to detect these three variants were reported to be >90% [317] …”

“…Compared to qRT-PCR, nucleic acid sequencing is said to be a more objective and unbiased test to detect the SARS-CoV-2 variant because the test user does not know about the virus information before the test [296] . Besides, some studies reported the use of qRT-PCR to screen for the presence of a specific target mutation, followed by nucleic acid sequencing tests like Sanger sequencing to confirm the mutation [309] , [310] . This approach could help to speed up test time and reduce test cost as there is no need to run whole-genome sequencing of the virus to confirm its identity [310] .…”

“…Besides, some studies reported the use of qRT-PCR to screen for the presence of a specific target mutation, followed by nucleic acid sequencing tests like Sanger sequencing to confirm the mutation [309] , [310] . This approach could help to speed up test time and reduce test cost as there is no need to run whole-genome sequencing of the virus to confirm its identity [310] . On contrary, SNP genotyping was also reported to be employed in detecting different SARS-CoV-2 variants and this assay was found to have the ability (>62%) to distinguish two virus variants of different genotypes [311] .…”

Possible link between higher transmissibility of B.1.617 and B.1.1.7 variants of SARS-CoV-2 and increased structural stability of its spike protein and hACE2 affinity

“…The Lineage B.1.1.7 or 20I/501Y.V1 was detected in the United Kingdom in October 2020. This variant has increased the transmissibility by 40-80%, which has been partially correlated with N501Y mutation in Receptor Binding Domain (RBD) of Spike protein 15 (Figure 1A). In December 2020, B.1.351 was detected in the South African population, which could infect more younger people and had three primary 4 mutations in the RBD of Spike protein, namely, N501Y, K417N and E484K 16 .…”

Monitoring SARS-CoV-2 Populations in Wastewater by Amplicon Sequencing and Using the Novel Program SAM Refiner

“…However, early in the SARS-CoV-2 pandemic, wastewater was used to track community levels and spread of SARS-CoV-2 by Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) methods [ 4 , 5 ]. Investigators have also used high-throughput sequencing on wastewater samples to obtain full or partial SARS-CoV-2 genomic sequences which have been used for metagenomic and epidemiologic analysis [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Sequences identified in wastewater samples may reflect known lineages as well as lineages not reported from clinical samples.…”