Identification of the SARS-CoV-2 Delta variant C22995A using a high-resolution melting curve RT-FRET-PCR

Abstract

Knowledge of SARS-CoV-2 variants is essential for formulating effective control policies. Currently, variants are only identified in relatively small percentages of cases as the required genome sequencing is expensive, time-consuming, and not always available. In countries with facilities to sequence the SARS-CoV-2, the Delta variant currently predominates. Elsewhere, the prevalence of the Delta variant is unclear. To avoid the need for sequencing, we investigated a RT-FRET-PCR that could detect all SARS-CoV-2 strains and simultaneously identify the Delta variant. The established Delta RT-FRET-PCR was performed on reference SARS-CoV-2 strains, and human nasal swab samples positive for the Delta and non-Delta strains. The Delta RT-FRET-PCR established in this study detected as few as ten copies of the DNA target and 100 copies of RNA target per reaction. Melting points of products obtained with SARS-CoV-2 Delta variants (around 56.1°C) were consistently higher than products obtained with non-Delta strains (around 52.5°C). The Delta RT-FRET-PCR can be used to diagnose COVID-19 patients and simultaneously identify if they are infected with the Delta variant. The Delta RT-FRET-PCR can be performed with all major thermocycler brands meaning data on Delta variant can now be readily generated in diagnostic laboratories worldwide.

SARS-CoV-2 Variants Identification: Overview of Molecular Existing Methods

“…Barua et al developed a reverse transcription fluorescence resonance energy transferpolymerase chain reaction (RT-FRET-PCR) designed to identify the T478K mutation (present in 99.73% of the Delta variant) that can be used both to diagnose COVID-19 patients and simultaneously identify if they are infected with the Delta variant; but the Delta variant is just one of several SARS-CoV-2 variants, so a continuous monitoring of strains will still be necessary [120].…”

Absence of SARS-CoV-2 in a captive white-tailed deer population in Alabama, USA

“…The High-Pure PCR Template Preparation Kit (Roche Diagnostics, Indianapolis, IN, USA) was used to extract total nucleic acids from nasopharyngeal and rectal swabs according to the manufacturer’s instructions and described previously [ 17 ]. SARS-CoV-2 Reverse-Transcription FRET-PCR was performed as described [ 18 ]. Genomic RNA of two SARS-CoV-2 viruses from the American Type Culture Collection (ATCC) (2019-nCOV/USA-WA1/2020; 201/501Y.V1) served as controls and quantitative standards.…”

Harringtonine has the effects of double blocking SARS-CoV-2 membrane fusion

“…Repeated epidemics are caused by the constant mutation of the SARS-CoV-2. The delta mutant strain was found in India as early as October 2020 18,19 rapidly spreading and became a major concern in the containment of the pandemic 20,21 . On November 9, 2021, the first Omicron sequence available was from a specimen collected in Botswana.…”