Genotyping of the Major SARS-CoV-2 Clade by Short-Amplicon High-Resolution Melting (SA-HRM) Analysis

Díaz-García, Héctor; Guzmán-Ortiz, Ana Laura; Ángeles-Floriano, Tania; Parra-Ortega, Israel; López-Martı́nez, Briceida; Martínez-Saucedo, Mirna; Aquino‐Jarquín, Guillermo; Sánchez-Urbina, Rocío; Quezada, Héctor; Granados-Riverón, Javier T.

doi:10.3390/genes12040531

Cited by 14 publications

(13 citation statements)

References 30 publications

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“…Furthermore, the qPCR-HRM method was proposed and is capable of detecting the D614G mutation by reliably distinguishing the D614 and G614 variants. A similar study examining the main clades of SARS-CoV-2 also proved that HRM analysis can effectively differentiate between variants of SARS-CoV-2 [ 18 ]. The other study described that HRM analysis also can rapidly differentiate Influenza A virus subtypes [ 28 ].…”

Section: Discussionmentioning

confidence: 93%

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

Gazali¹,

Nuhamunada²,

Nabilla³

et al. 2021

Heliyon

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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.

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Section: Discussionmentioning

confidence: 93%

“…Another possible and reliable method for detecting SNP is real-time PCR and high-resolution melting curve analysis (HRM) [ 12 ]. In addition, it has been reported that the HRM analysis accurately distinguishes between GR and non-GR clades of SARS-CoV-2 [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

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

Gazali¹,

Nuhamunada²,

Nabilla³

et al. 2021

Heliyon

View full text Add to dashboard Cite

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“…Of note, the estimated cost for the HRM-RTqPCR assay validated herein was US$ 10 per sample, a cost considerably lower than any commercial kit available for the molecular diagnostic testing of COVID-19 including TaqMan-based systems and conventional DNA sequencing used to identify SARS-CoV-2 variants. Altogether, SYBR-Green and HRM-based methodologies are emerging in the literature as promising alternatives to the molecular diagnostics of COVID-19 and identification of SARS-CoV-2 variants of concern [22][23][24][25].…”

Section: Plos Onementioning

confidence: 99%

Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting

Ferreira¹,

Silva-Gomes²,

Coelho

et al. 2021

PLoS ONE

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The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

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“…The majority of the targeted assays rely on PCR amplification of a genomic region followed by amplicon Sanger sequencing ( Cabral et al, 2021 , Sanger Sequencing Solutions for SARS-CoV-2 Research - CA 2021 ). Other assays are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) using TaqMan probes targeting specific SNPs ( Bier et al, 2021 ), amplicon melting curve analysis, or high range melting (HRM) analysis ( Banada et al, 2021 , Diaz-Garcia et al, 2021 ). These targeted assays are limited to currently known SNPs related to VOC/VOI and do not have the potential for rapid identification of new mutations, especially during a pandemic where turnaround times are a critical factor for molecular surveillance.…”

Section: Introductionmentioning

confidence: 99%