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.

中文翻译：

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使用双泡引物改进了 SARS-CoV-2 PCR 检测和基因分型。

描述了一种改进 RT-PCR 的新方法。它基于设计用于形成受控茎环和同二聚体构型的引物，因此被称为“双泡”引物。引物包含用于高效 RT-PCR 的三个主要区域：允许逆转录的 3' 短突出端、用于热启动的茎区域和用于 PCR 扩增的模板特异性区域。作为原理证明，使用GAPDH、SARS-CoV-2 合成 RNA 和 SARS-CoV-2 病毒阳性鼻咽拭子作为模板。此外，这些引物还用于阳性确认鼻咽拭子中的 N501Y 突变。有证据表明，双泡引物在 RT-PCR 扩增方面提供了快速、特异、稳健且经济高效的改进，可用于一般基因表达检测，特别是 SARS-CoV-2 的诊断检测和基因分型。