Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants

Abstract

Reverse transcription quantitative PCR (RT-qPCR) assays are gold standard in diagnosing SARS-CoV-2 infection and play a major role in viral subtyping for rapid detection and monitoring of important mutations, containing the spread of new virus variants. We wanted to compare RT-qPCR melting curve analysis assays to Sanger Sequencing for detection of variants within the SARS-CoV-2 spike glycoprotein and examined their sensitivity and specificity. Samples positive for SARS-CoV-2 (n = 663 + 82) were subtyped using both Sanger sequencing and five RT-qPCR melting curve analysis assays specific for the mutations N501Y, P681H, E484K, K417N/T, and N439K. The results of the two methods were compared. The training cohort and the clinical validation cohort showed equally, or significantly better sensitivity of the assays compared to the Sanger sequencing. The agreement of the Sanger sequencing and the assays ranged from 92.6 to 100% for the training cohort and 99.4–100% for the clinical validation. The sensitivity, specificity, and turn-around time of the RT-qPCR melting curve analysis assays are well-suited for clinical monitoring of VOCs, making the assays an important tool in contact tracing and risk stratification. Furthermore, the assays were able to indicate the presence of new mutations in the complementary sequence to the mutation-specific probes.

Rapid and Accurate Detection of the SARS-CoV-2 Omicron Variant with a CRISPR-Cas12a Reaction in the RT-qPCR Pot

“…In less than 15 min, this supplementary reaction was able to provide a suitable indication, which saves valuable time compared to sequencing and allows information to be provided to the patient at diagnosis. Intriguingly, this procedure may even be complementary to those RT-qPCR-based methods developed for virus variant determination − to double-check the results or detect additional mutations of interest.…”

“…To overcome this issue, we have designed appropriate primers and probes to run RT-qPCRs aimed at detecting specific SARS-CoV-2 variants. These approaches comprise target amplification failure, allele-specific amplification and probing, , and melting curve analysis . Yet, these methods are not fully insensitive to spurious amplifications, and they often require extensive screening of concentrations and annealing temperatures.…”

“…These approaches comprise target amplification failure, 8 allele-specific amplification and probing, 9 , 10 and melting curve analysis. 11 Yet, these methods are not fully insensitive to spurious amplifications, and they often require extensive screening of concentrations and annealing temperatures. Here, we propose a novel and simple approach to detect virus variants with ultraspecificity by means of a CRISPR-Cas12a assay 12 on top of the routine RT-qPCR ( Figure 1 ; CRISPR stands for clustered regularly interspaced short palindromic repeats).…”

Two‐Stage Mixed‐Dye‐Based Isothermal Amplification with Ribonuclease‐Cleavable Enhanced Probes for Dual‐Visualization Detection of SARS‐CoV‐2 Variants of Interest

“…[7] Resembling NGS, this routine method has to be finished using a sophisticated sequencer. [8] Apart from Sanger sequencing, other strategies such as TaqMan probe, [9] mass spectrometry, [10] and melting curve analysis [11] are also interfaced with RT-PCR for SARS-CoV-2 variants detection. Unfortunately, the core RT-PCR technology relies on benchtop thermal cycling equipment and requires approximately two-hour assay time, unable to reach the goal of rapidly detecting SARS-CoV-2 variants detection.…”