Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR

Autor: Chunyan Niu, Xia Wang, Yunhua Gao, Xiaoting Qiao, Jie Xie, Yongzhuo Zhang, Di Wang, Lianhua Dong
Rok vydání: 2022
Předmět:
Zdroj: Analytical and bioanalytical chemistry. 414(23)
ISSN: 1618-2650
Popis: The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 505 million confirmed cases, including over 6 million deaths. Reference materials (RMs) of SARS-CoV-2 RNA played a crucial role in performance evaluation and quality control of testing laboratories. As the potential primary characterization method of RMs, reverse transcription digital PCR (RT-dPCR) measures the copy number of RNA, but the accuracy of reverse transcription (RT) efficiency has yet to be confirmed. This study established a method of enzymatic digestion followed by isotope dilution mass spectrometry (IDMS), which does not require an RT reaction, to quantify in vitro-transcribed SARS-CoV-2 RNA. RNA was digested to nucleotide monophosphate (NMP) within 15 min and analyzed by IDMS within 5 min. The consistency among the results of four different NMPs demonstrated the reliability of the proposed method. Compared to IDMS, the quantitative result of RT-dPCR turned out to be about 10% lower, possibly attributed to the incompleteness of the reverse transcription process. Therefore, the proposed approach could be valuable and reliable for quantifying RNA molecules and evaluating the RT efficiency of RT-based methods.
Databáze: OpenAIRE
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