A rapid and low-cost protocol for the detection of B.1.1.7 lineage of SARS-CoV-2 by using SYBR Green-based RT-qPCR

Abstract

Background: The new SARS-CoV-2 variant VOC (202012/01), identified recently in the United Kingdom (UK), exhibits a higher transmissibility rate compared to other variants, and a reproductive number 0.4 higher. In the UK, scientists were able to identify the increase of this new variant through the rise of false negative results for the spike (S) target using a three-target RT-PCR assay (TaqPath kit).

Methods: To control and study the current coronavirus pandemic, it is important to develop a rapid and low-cost molecular test to identify the aforementioned variant. In this work, we designed primer sets specific to the VOC (202012/01) to be used by SYBR Green-based RT-PCR. These primers were specifically designed to confirm the deletion mutations Δ69/Δ70 in the spike and the Δ106/Δ107/Δ108 in the NSP6 gene. We studied 20 samples from positive patients, detected by using the Applied Biosystems TaqPath RT-PCR COVID-19 kit (Thermo Fisher Scientific, Waltham, USA) that included the ORF1ab, S, and N gene targets. 16 samples displayed an S-negative profile (negative for S target and positive for N and ORF1ab targets) and four samples with S, N and ORF1ab positive profile.

Results: Our results emphasized that all S-negative samples harbored the mutations Δ69/Δ70 and Δ106/Δ107/Δ108. This protocol could be used as a second test to confirm the diagnosis in patients who were already positive to COVID-19 but showed false negative results for S-gene.

Conclusions: This technique may allow to identify patients carrying the VOC (202012/01) or a closely related variant, in case of shortage in sequencing.

Keywords: B.1.1.7 lineage; COVID-19 pandemic; SARS CoV-2; SYBR Green-based RT-PCR; VOC (202012/01).

Fig. 1

The Localization of the selected primers. Deleted nucleotides are written in bold

Fig. 2

Real time PCR results, from SYBR Green-Based assay, with primers targeting the non-mutated N, Spike and NSP6 genes in S-positive and S-negative samples (N-gene is used as a positive control). Panel A show the amplification curves of the targeted regions in N, S and NSP6 genes in the S-positive samples. Panel B show the amplification curves of the targeted region in N gene in the S-negative samples. Panel C and D show the melting curves of the targeted regions in S-positive and S-negative samples, respectively. In panel D only one melting peak in S-negative samples, corresponding to the N gene

Fig. 3

SYBR Green-based PCR with primers targeting the mutated Spike del 69/70 genes in S-negative samples (N-gene is used as a positive control). Panel A show the amplification curves of the targeted regions in N, and Spike genes in the S-negative samples using three primer sets corresponding to N, Spike WT and Spike del 69/70. Panel B show the melting curves for the products amplified in S-negative samples by N and Spike del 69/70 primers only