The Impact of Accumulated Mutations in SARS-CoV-2 Variants on the qPCR Detection Efficiency

Abstract

SARS-CoV-2 is evolving with mutations throughout the genome all the time and a number of major variants emerged, including several variants of concern (VOC), such as Delta and Omicron variants. In this study, we demonstrated that mutations in the regions corresponding to the sequences of the probes and 3’-end of primers have a significant impact on qPCR detection efficiency. We also found that the G28916T mutation of the N gene accounts for 78.78% sequenced genomes of Delta variant. It was found that detection sensitivity of G28916T mutant was 2.35 and 1.74 times less than that of the wt sequence and detection limit was reduced from 1 copy/μl to 10 copies/μl for the commercially available CP3 and CP4 primer/probe sets. These results indicate that the detection probes and primers should be optimized to keep maximal detection efficiency in response to the emergence of new variants.

Performance Evaluation of Developed Bangasure™ Multiplex rRT-PCR Assay for SARS-CoV-2 Detection in Bangladesh: A Blinded Observational Study at Two Different Sites

“…Two groups of primer-probe sets (Charité/Berlin and CDC designed Primer probe sets) which are recognized globally and used in many diagnostic assays was primarily chosen to be evaluated. Evaluation of these primer probes to find an optimum combination was conducted by thoroughly reviewing the literature [ 25 , 26 , 27 , 28 , 29 , 30 ]. WHO recommended the Charité/Berlin assay which detects two viral targets E and RdRp gene [ 16 ].…”

“…On the other hand, comparing the Ct values and analytical sensitivity of N1 and N2 primer-probe sets, N2 performed slightly better than the N1 primer-probe set [ 28 ]. Moreover, the N1 probe has a single nucleotide mismatch in more than 98% of Omicron variants which is a variant of concern and the most predominant SARS-CoV-2 variant in the world right now [ 26 ]. After analyzing all these facts and following the recommendation of Nalla et al, 2020, the E primer-probe of Charité/Berlin and N2 primer-probe designed by CDC was chosen for in-house assay [ 28 ].…”

SARS-CoV-2: An Overview of the Genetic Profile and Vaccine Effectiveness of the Five Variants of Concern

“…The qPCR method used for detecting SARS-CoV-2 virus is a technique where mainly the N and S genes of the virus are identified. Given that these genes have a high probability of mutation, the accuracy of the tests may be affected [ 114 ]. Regarding the spike protein, it has unique nucleotide sequences for SARS-CoV-2 minimizing cross-reactivity and false-positive results in the presence of other types of Coronaviruses.…”

Host tRNA-Derived RNAs Target the 3′Untranslated Region of SARS-CoV-2

“…Each variant that posed a significant risk to public health was termed a variant of concern (VOC) by the World Health Organization. These VOCs impact the need for changes in vaccines and testing protocols and pose challenges to the development of novel therapeutic interventions [5][6][7]. The persistence of new RNA virus outbreaks like SARS-CoV-2 coupled with the possibility of an increase in frequency of new epidemics highlights a heightened need for novel antiviral therapeutic targets [8].…”