Detection of SARS-CoV-2 spike protein D614G mutation by qPCR-HRM analysis

Abstract

Objectives Monitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase quantitative polymerase chain reaction - high resolution melting analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome. Methods Six SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods. Results The results showed that the melting temperature (T m ) of the D614 variant was 79.39 ± 0.03°C, which was slightly lower than the T m of the G614 variant (79.62 ± 0.015°C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing. Conclusions This study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation.

Rapid and Affordable High Throughput Screening of SARS-CoV-2 Variants Using Denaturing High-Performance Liquid Chromatography Analysis

“…does not require complicated procedures for establishing or maintaining analytical performance as designing and validating increasing number of probes, or cumbersome technical replicatesOther sequence non-specific methods, such as High-Resolution Melting (HRM) may be valid alternatives. Nonetheless, HRM has been limited by inherently shorter amplicons and a longer running time (45)(46)(47)(48)(49)(50). Furthermore, HRM approaches are greatly limited to discrete mutations targeted to achieve consistency and reliability in performance (45)(46)(47)(48)(49)(50) In conclusion, the advantages for the healthcare systems of the flexible, cost-effective streamlined approach herein described, which integrates DHPLC with Sanger sequencing and, eventually, NGS should be considered as valuable alternative for variant screening in the genomic surveillance.…”

“…Nonetheless, HRM has been limited by inherently shorter amplicons and a longer running time (45)(46)(47)(48)(49)(50). Furthermore, HRM approaches are greatly limited to discrete mutations targeted to achieve consistency and reliability in performance (45)(46)(47)(48)(49)(50) In conclusion, the advantages for the healthcare systems of the flexible, cost-effective streamlined approach herein described, which integrates DHPLC with Sanger sequencing and, eventually, NGS should be considered as valuable alternative for variant screening in the genomic surveillance.…”

Real-time allelic assays of SARS-CoV-2 variants to enhance sewage surveillance

“…Several PCR-based approaches have been developed for the rapid discrimination of SARS-CoV-2 variants in clinical samples, such as the RT-PCR melting temperature assay ( Banada et al, 2021a ; Banada et al, 2021b ) and multiplex qPCR target failure screening ( Vogels et al, 2021 ). The former method requires a clear difference in the annealing temperatures between the target mutation sequence and its opposite genotype, which could be affected by any additional mutations located in the target sequence ( Gazali et al, 2021 ). The multiplex qPCR target failure screening is based on deletions in the genome of SARS-CoV-2, and does not have good sensitivity and specificity since the rapid emergence of new mutations is the major concern.…”

Machine Learning-Assisted Real-Time Polymerase Chain Reaction and High-Resolution Melt Analysis for SARS-CoV-2 Variant Identification

“…Due to high sensitivity, HRM has been extensively used for single nucleotide polymorphism (SNP) detection. Besides, numerous studies have utilized HRM in diverse applications, e.g., detection of genetic diseases, − mutations, bacteria, − and genotyping. − Recently, HRM has also been employed for SARS-CoV-2 variant identification − and mutation detection . These studies have shown the feasibility of HRM as a practical genotyping tool for SARS-CoV-2 variant determination.…”

“…23−29 Recently, HRM has also been employed for SARS-CoV-2 variant identification 30−32 and mutation detection. 33 These studies have shown the feasibility of HRM as a practical genotyping tool for SARS-CoV-2 variant determination. The use of HRM analysis to detect variants complements the existing detection method of SARS-CoV-2 by real-time reverse transcription PCR (qRT-PCR).…”