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Rapid Detection of Predominant SARS-CoV-2 Variants Using EvaGreen® Dye in Multiplex High-Resolution Melting Analysis

Since the onset of the SARS-CoV-2 global pandemic in late 2019, mutated variants of concern (VOCs) have emerged that elicit the need for more sensitive surveillance and efficient detection. This is especially important for the Delta and Omicron variants, as these two are highly transmissible and contain multiple S gene mutations. Such factors underscore the importance for timely detection beyond the current genomic and Sanger sequencing methods that are typically both time consuming and costly.

In a May 2023 publication in Microbiology Spectrum, L Sun et al. developed a high-resolution melting (HRM®) assay to effectively detect SARS-CoV-2 variants without using time consuming and costly sequencing methods. This technique integrated multiple processes into a single-tube reaction and utilized Biotium’s EvaGreen® Dye to target eight key mutation sites, including those in the receptor binding domain (RBD) associated with enhanced infectivity and immune escape. Biotium’s EvaGreen® Dye was purposefully chosen by the researchers for its sensitivity, reliability, and cost-effectiveness. After analyzing 324 clinical samples of nasopharyngeal swabs and sputum through reverse transcription-quantitative PCR (qRT-PCR) and the multiplex HRM® method, both methods detected 48 samples positive for SARS-CoV-2, with strains of Delta, Omicron-BA.1, Omicron-BA.2, and Omicron-BA.5. The multiplex HRM® analysis had a limit of detection (LOD) lower than 10 copies/reaction for various targets and exhibited no cross-reactivity with other pathogens, achieving a robust 97.9% concordance rate with standard Sanger sequencing. The multiplex HRM® analysis clearly identified differentiated mutations in approximately two hours. The authors found that multiplex HRM® to be more reliable, economical, rapid, and expandable than sequencing for monitoring new viral variants as they emerge. With ever-evolving SARS-CoV-2 variants, this method may empower researchers and healthcare professionals with a convenient and faster alternative for timely diagnosis and surveillance.

Figure 1 shows the experimental workflow and research design. Specifically, the multiplex HRM® reaction required 10 μL 2× reaction mix (Invitrogen, Carlsbad, CA, USA), 1 μL 20× EvaGreen® fluorescent dye (Biotium, Fremont, CA, USA), 1 μL SuperScript III RT/Platinum Taq mix (Invitrogen), the corresponding volumes of primer pools for each assay, 2 μL of the RNA template, and nuclease-free water, rendering a total reaction volume of 20 μL. The multiplex HRM® reaction conditions were as follows: reverse transcription-PCR for 30 min at 55°C; PCR activation for 2 min at 95°C; 40 cycles of amplification for 30 s at 94°C and 15 s at 53°C and 15 s of signal collection at 68°C; a final HRM® step of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s, and continuous signal collection from 60 to 95°C at an increment rate of 0.025°C/s; and then cooling at 60°C. One-step multiplex HRM® was performed using the Applied Biosystems QuantStudio 6 Flex real-time PCR instrument with a fast 96-well block and HRM® capacity (Applied Biosystems, Inc., Waltham, MA, USA). The melting curve and Tm values were analyzed using the HRM® module in QuantStudio real-time PCR software v1.2. Credit: Sun, L. et al (2023). reproduced under the Creative Commons license.

Full Citation

Sun, L., Wang, L., Zhang, C. et al (2023). Rapid detection of predominant SARS-COV-2 variants using multiplex high-resolution melting analysis. Microbiology Spectrum (2023). 

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