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. 2021 Sep;93(9):5588-5593.
doi: 10.1002/jmv.27139. Epub 2021 Jun 22.

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

Subarna Barua  1 , Monirul Hoque  1 , Patrick J Kelly  2 , Jianfa Bai  3 , Gregg Hanzlicek  3 , Lance Noll  3 , Heather Walz  1   4 , Calvin Johnson  1 , Constantinos Kyriakis  1 , Chengming Wang  1
Affiliations

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

Subarna Barua et al. J Med Virol. 2021 Sep.

Abstract

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.

Keywords: FRET-PCR; SARS-CoV-2; high-resolution melting curve analysis; mutations.

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Conflict of interest statement

The authors declare that there are no conflict of interests.

Figures

Figure 1
Figure 1
Melting temperature (T m) analysis of SARS‐CoV‐2 controls and feline and human isolates with an RT‐FRET‐PCR for the A23403G mutation. The 6‐FAM probe designed to match exactly with the SARS‐CoV‐2 control without the mutation (2019‐nCOV/USA‐WA1/2020) had a T m of 63.1°C. This was irrespective of copy number. With the SARS‐CoV‐2 control that had the mutation (201/501Y.V1) there was an A to G mismatch with the probe (chromas graph C) that resulted in a lower T m of 58.2°C. (B) RT FRET‐PCRs of the clinical samples from a cat and people all had a T m of around 58.2°C indicating the presence of the A23403G mutation. 6‐FAM, 6‐carboxyfluorescein; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus‐2
Figure 2
Figure 2
Melting temperature (T m) analysis of SARS‐CoV‐2 controls and feline and human isolates with an RT‐FRET‐PCR for the C14408T mutation. The 6‐FAM probe designed to match exactly with the SARS‐CoV‐2 control without the mutation (201/501Y.V1) had a T m of 57.7°C. This was irrespective of copy number. With the SARS‐CoV‐2 control that had the mutation (201/501Y.V1) there was an A to C mismatch with the probe (chromas graph C) that resulted in a lower T m of 54.3°C. (B) RT FRET‐PCRs of the clinical samples from a cat and people all had a T m of around 54.3°C with this RT FRET‐PCR indicating the presence of the C14408T mutation. SARS‐CoV‐2, severe acute respiratory syndrome coronavirus‐2
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