Surveillance of SARS-CoV-2 lineage B.1.1.7 in Slovakia using a novel, multiplexed RT-qPCR assay

Abstract

The emergence of a novel SARS-CoV-2 B.1.1.7 variant sparked global alarm due to increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect B.1.1.7 include sequencing and RT-qPCR tests containing a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant. Since many countries lack genomic surveillance programs and failed assays detect unrelated variants containing similar mutations as B.1.1.7, we used allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop an RT-qPCR test that accurately and rapidly differentiates B.1.1.7 from other SARS-CoV-2 variants. We validated the test on 106 clinical samples with lineage status confirmed by sequencing and conducted a country-wide surveillance study of B.1.1.7 prevalence in Slovakia. Our multiplexed RT-qPCR test showed 97% clinical sensitivity and retesting 6,886 SARS-CoV-2 positive samples obtained during three campaigns performed within one month, revealed pervasive spread of B.1.1.7 with an average prevalence of 82%. Labs can easily implement this test to rapidly scale B.1.1.7 surveillance efforts and it is particularly useful in countries with high prevalence of variants possessing only the ΔH69/ΔV70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.

Genomic Surveillance Enables the Identification of Co-infections With Multiple SARS-CoV-2 Lineages in Equatorial Guinea

“…A cost-efficient alternative to whole genome sequencing are multiplex reverse transcription quantitative polymerase chain reactions (RT-qPCR) assays which detect relevant mutations associated with SARS-CoV-2 variants of concern (VOC) (2)(3)(4). Mutation-specific assays can complement genomic surveillance programs, especially in settings where widespread sequencing capabilities are not available.…”

Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols

“…Rapid, cost-effective, and near real-time genome sequencing of the SARS-CoV-2 variants combined with epidemiological data provides an important resource not only for understanding the virus transmission, its genetic alterations and evolution, but also for making the policy decisions in combating the pandemic [5]. Monitoring sequence diversification plays an essential role in continual refinement of molecular diagnostics (e.g., redesigning the primers for nucleic acid amplification techniques [6] or development of screening tools for variants of concerns (VoC) and those evading the immune response [7,8]). This underscores the importance of genomic epidemiology, although the elucidation of direct links between particular mutation (s) and the virus spreading or clinical implications still represents a challenging task [9][10][11][12][13][14][15][16][17][18][19].…”

Evaluation of a Rapid and Accessible Reverse Transcription-Quantitative PCR Approach for SARS-CoV-2 Variant of Concern Identification

“…Several groups previously described similar approaches to SARS-CoV-2 variant determination by RT-qPCR and digital droplet RT-PCR, particularly for the spike del69–70, E484K, and N501Y mutations ( 32 – 39 ). Some of those assays included additional mutation sites that were not in our study, such as spike del144 or open reading frame 1a (ORF1a) Δ3675–3677 ( 32 , 38 ). Those earlier assays, published prior to the rise of Delta, primarily targeted VOCs Alpha, Beta, and Gamma.…”