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

• Published in: Analytical and bioanalytical chemistry Vol. 414; no. 23; pp. 6771 - 6777
• Main Authors: Niu, Chunyan, Wang, Xia, Gao, Yunhua, Qiao, Xiaoting, Xie, Jie, Zhang, Yongzhuo, Wang, Di, Dong, Lianhua
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2022; Springer; Springer Nature B.V
• Subjects: Analysis; Analytical Chemistry; Biochemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Copy number; Coronaviruses; COVID-19; Dilution; Efficiency; Food Science; Genetic transcription; Laboratory Medicine; Mass spectrometry; Mass spectroscopy; Methods; Monitoring/Environmental Analysis; Nucleotides; Performance evaluation; Polymerase chain reaction; Quality control; Research Paper; Reverse transcription; Ribonucleic acid; RNA; Scientific imaging; Severe acute respiratory syndrome coronavirus 2; Spectroscopy; Testing laboratories; Viral diseases; China; Reverse transcription; SARS-CoV-2; Digital PCR; IDMS
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-022-04238-6

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. Graphical abstract