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Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers.

ABSTRACT: A new approach for improved RT-PCR is described. It is based on primers designed to form controlled stem-loop and homodimer configurations, hence the name 'double-bubble' primers. The primers contain three main regions for efficient RT-PCR: a 3' short overhang to allow reverse transcription, a stem region for hot start and a template-specific region for PCR amplification. As proof of principle, GAPDH, SARS-CoV-2 synthetic RNA and SARS-CoV-2 virus-positive nasopharyngeal swabs were used as templates. Additionally, these primers were used to positively confirm the N501Y mutation from nasopharyngeal swabs. Evidence is presented that the double-bubble primers offer fast, specific, robust and cost-effective improvement in RT-PCR amplification for detection of gene expression in general and for diagnostic detection and genotyping of SARS-CoV-2 in particular.

SUBMITTER: Ailenberg M

PROVIDER: S-EPMC8544493 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers.

Ailenberg Menachem M Kapus Andras A Rotstein Ori D OD

BioTechniques 20210914 6

A new approach for improved RT-PCR is described. It is based on primers designed to form controlled stem-loop and homodimer configurations, hence the name 'double-bubble' primers. The primers contain three main regions for efficient RT-PCR: a 3' short overhang to allow reverse transcription, a stem region for hot start and a template-specific region for PCR amplification. As proof of principle, <i>GAPDH</i>, SARS-CoV-2 synthetic RNA and SARS-CoV-2 virus-positive nasopharyngeal swabs were used as ...[more]

PMID: 34519222

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