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
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 Delta 69/Delta 70 in the spike and the Delta 106/Delta 107/Delta 108 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 Delta 69/Delta 70 and Delta 106/Delta 107/Delta 108. 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.