期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：35
DOI：10.1073/pnas.2100347118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
We report a rapid COVID-19 assay that gives a sample-to-signal time of under 10 min. The current gold-standard COVID-19 assay uses PCR, where strands of DNA are copied (amplified) many times to generate a read-out signal. However, as the virus genome is RNA, first conversion into DNA is required using reverse transcription (RT) before amplification. While just as sensitive, our assay is faster because 1) we have designed a method for generating DNA (the trigger strand) from RNA, bypassing the lengthy RT step, and 2) a quicker amplification process than PCR, called exponential amplification reaction (EXPAR), is used to amplify the trigger. This methodology could ultimately be applied to any RNA-based assay, including the detection of other infectious agents.
A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription–free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.
