key: cord-0816483-cam5mq5c
authors: Barua, Subarna; Hoque, Monirul; Kelly, Patrick J.; Bai, Jianfa; Hanzlicek, Gregg; Noll, Lance; Walz, Heather; Johnson, Calvin; Kyriakis, Constantinos; Wang, Chengming
title: High‐resolution melting curve FRET‐PCR rapidly identifies SARS‐CoV‐2 mutations
date: 2021-06-22
journal: J Med Virol
DOI: 10.1002/jmv.27139
sha: 1f5c10abc9dda491e883e393c8fd194f9186b643
doc_id: 816483
cord_uid: cam5mq5c

Reverse transcription fluorescence resonance energy transfer‐polymerase chain reaction (FRET‐PCRs) were designed against the two most common mutations in severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) (A23403G in the spike protein; C14408T in the RNA‐dependent RNA polymerase). Based on high‐resolution melting curve analysis, the reverse transcription (RT) FRET‐PCRs identified the mutations in american type culture collection control viruses, and feline and human clinical samples. All major makes of PCR machines can perform melting curve analysis and thus further specifically designed FRET‐PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available.

to be categorized into eight clades 1 and six major lineages. 2 Some emerging SARS-CoV-2 variants may have increased potential for transmissibility and virulence and lowered protection from vaccines. [3] [4] [5] [6] [7] [8] Surveillance for variants and their spread is important in understanding the dynamics of the COVID-19 pandemic and in developing effective control policies. Screening for variants, however, is generally infrequent as it requires genome sequencing which is expensive, time-consuming, and not readily available in most countries. 9 Comparison of sequences in global initiative on sharing avian influenza data (GISAID) and reported cases from the six countries most affected by COVID-19 reveals genotyping was only performed on under 1% of cases in the United States, Brazil, India, France, Russia, Italy, and South Africa, and 8.3% in the UK (Table S1 ).

As it is practically impossible for even the most advanced countries to sequence all positive samples, it would be very useful if tests were available which could be readily used by la- diagnostic laboratories around the world to monitor variants rapidly and conveniently, and thereby implement more targeted and appropriate control programs.

The A23403G and the C14408T mutations are the most common mutations from the original Wuhan strain that persists in almost all variants today. 9 They are present in all variants of interest (VOI) and variants of concern (VOC) determined by the CDC 10 and reported to be the most common in the United States and globally. 11 We confirmed this by analyzing all available highquality SARS-CoV-2 sequences from GISAID (https://www.gisaid. 

Representative sequences around the mutations were aligned, and upstream and downstream primers and probes were designed to amplify and detect all SARS-CoV-2. The 6-carboxyfluorescein (6-FAM)-labeled probes were further designed to contain the unique A23403G or C14408T mutation ( Table 2 ). The 6-FAM probe was 3ʹ The melting curve which assesses the dissociation of the PCR products and labeled probes was determined by monitoring the fluorescence from 35°C to 75°C with a temperature transition rate of 0.2°C per second. The first derivatives of F2/F1 were evaluated to determine the T m of the probe (Figures 1 and 2) .

Nucleotide mismatches between the 6-FAM-probes and the SARS-CoV-2 variants result in distinct T m values. FRET-PCRs can be readily used for active surveillance and screening for mutations and variants, thereby reducing requirements for sequencing. It can be also used for large-scale retrospective molecular epidemiology studies of SARS-CoV-2 and its variants worldwide.

In conclusion, we have shown highly sensitive RT FRET-PCRs can be developed to detect SARS-CoV-2 infections and to determine whether specific mutations are present. This highly specific and readily available platform should be able to be readily and rapidly adapted to monitor the presence of other mutations and associated variants that are of concern in countries around the world. This technique will greatly facilitate the monitoring of the origins and spread of mutations in variants in the COVID-19 pandemic and more readily provide data that can be used for public health intervention programs.

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How to cite this article

High-resolution melting curve FRET-PCR rapidly identifies SARS-CoV-2 mutations

The authors declare that there are no conflict of interests. All authors have read and approved the manuscript.

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