Abstract
COVID-19 pandemic highlighted the demand for the fast and reliable detection of viral RNA. Although various methods for RNA amplification and detection have been proposed, some limitations, including those caused by reverse transcription (RT), need to be overcome. Here, we report on the direct detection of specific RNA by conventional polymerase chain reaction (PCR) requiring no prior RT step. It was found that Hemo KlenTaq (HKTaq), which is posed as DNA-dependent DNA polymerase, possesses reverse transcriptase activity and provides reproducible amplification of RNA targets with an efficiency comparable to common RT-PCR. Using nasopharyngeal swab extracts from COVID-19-positive patients, the high reliability of SARS-CoV-2 detection based on HKTaq was demonstrated. The most accurate detection of specific targets are provided by nearby primers, which allow to determine RNA in solutions affected to multiple freeze-thaw cycles. HKTaq can be used for elaboration of simplified amplification techniques intended for the analysis of any specific RNA and requiring only one DNA polymerase.
Keywords: Degraded RNA; Nearby primers; Polymerase chain reaction; Reverse transcriptase activity; SARS-CoV-2; Viral RNA.
【저자키워드】 SARS-CoV-2, polymerase chain reaction, Viral RNA., Reverse transcriptase activity, Nearby primers, Degraded RNA, 【초록키워드】 COVID-19, pandemic, reliability, RT-PCR, RNA, Nasopharyngeal swab, amplification, SARS-CoV-2 detection, PCR, target, reverse transcription, Viral RNA, patients, Analysis, Efficiency, reverse transcriptase, Primers, transcriptase, DNA polymerase, limitations, polymerase chain, affected, caused, provided, can be used, provide, determine, overcome, demonstrated, comparable, freeze-thaw cycles, Nearby, 【제목키워드】 Viral RNA, DNA polymerase,