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 costeffective improvement in RT-PCR amplification for detection of gene expression in general and for diagnostic detection and genotyping of SARSCoV- 2 in particular. METHOD SUMMARY 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. It is fast, specific, robust and cost-effective. The method is applied to detect wild-type and mutated SARS-CoV-2 virus.