Limitation of Screening of Different Variants of SARS-CoV-2 by RT-PCR

Abstract

Since January 2021, the diffusion of the most propagated SARS-CoV-2 variants in France (UK variant 20I/501Y.V1 (lineage B.1.1.7), 20H/H501Y.V2 (lineage B.1.351) and 20J/H501Y.V3 (lineage P.1)) were urgently screened, needing a surveillance with an RT-PCR screening assay. In this study, we evaluated one RT-PCR kit for this screening (ID SARS-CoV-2/UK/SA Variant Triplex®, ID Solutions, Grabels, France) on 2207 nasopharyngeal samples that were positive for SARS-CoV-2. Using ID Solutions kit, 4.1% (92/2207) of samples were suspected to belonged to B.1.351 or P.1 variants. Next-generation sequencing that was performed on 67.4% (62/92) of these samples confirmed the presence of a B.1.351 variant in only 75.8% of the samples (47/62). Thirteen samples belonged to the UK variant (B.1.1.7), and two to A.27 with N501Y mutation. The thirteen with the UK variant presented one mutation in the S-gene, near the ΔH69/ΔV70 deletion (S71F or A67S), which impacted the detection of ΔH69/ΔV70 deletion. Using another screening kit (PKampVariantDetect SARS-CoV-2 RT-PCR combination 1 and 3® PerkinElmer, Waltham, MA, USA) on the misidentified samples, we observed that the two mutations, S71F or A67S, did not impact the detection of the UK variant. In conclusion, this study highlights the limitations of the screening strategy based on the detection of few mutations/deletions as well as it not being able to follow the virus evolution.

Evaluation of a commercial SARS-CoV-2 multiplex PCR genotyping assay for variant identification in resource-scarce settings

“…Furthermore, our results are consistent with similar studies that have recently evaluated Seegene Variant assays [21,22]. This observation reinforces the predictive value of commercial multiplex PCR genotyping assays in epidemiological surveillance and highlights the diagnostic utility this approach can play in supporting WGS in resource-scarce and under-represented settings, as has been shown in other studies [23][24][25][26][27][28][29]. Additionally, this strategy is rapid, reproducible, and technically easy.…”

“…Commercial multiplex PCR genotyping kits can be combined to maximize variant detection capacity and identify novel variants. There are a number of variant PCR assays which, when combined, allow for detection of multiple SAR-CoV-2 variants [21,22,28,29,33,34].…”

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform

“…Conversely, the accuracy of the most frequently used RT-qPCR for SARS-CoV-2 Marseille-4 variant detection, a point mutation in ORF1, with a specific probe is 93% ( Bedotto et al, 2021 ). Furthermore, the ability of RT-qPCR to differentiate mutations distinguishment is influenced by adjacent mutations, resulting in decreased accuracy ( Boudet et al, 2021 ; Zelyas et al, 2021 ). This issue illustrates a limitation of RT-qPCR for variant screening.…”

SARS-CoV-2 Variants of Concern (VOC) Alpha, Beta, Gamma, Delta, and Omicron coincident with consecutive pandemic waves in Pakistan

“…We have demonstrated that a PCR-based targeted approach can be used to monitor VOCs in a low-resource setting. However, this approach is unable to fully characterize multiple genetic variations of SARS-CoV-2 lineages and study viral evolution 26 . Furthermore, this method cannot identify minor and novel variants and several strains were 'undetermined'.…”