tetano
Editor, Senior Moderator
Anal Bioanal Chem
. 2022 Aug 9.
doi: 10.1007/s00216-022-04238-6. Online ahead of print.
Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR
Chunyan Niu[SUP] 1 [/SUP], Xia Wang[SUP] 1 [/SUP], Yunhua Gao[SUP] 1 [/SUP], Xiaoting Qiao[SUP] 1 [/SUP], Jie Xie[SUP] 1 [/SUP], Yongzhuo Zhang[SUP] 1 [/SUP], Di Wang[SUP] 1 [/SUP], Lianhua Dong[SUP] 2 [/SUP]
Affiliations
Abstract
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.
Keywords: Digital PCR; IDMS; Reverse transcription; SARS-CoV-2.
. 2022 Aug 9.
doi: 10.1007/s00216-022-04238-6. Online ahead of print.
Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR
Chunyan Niu[SUP] 1 [/SUP], Xia Wang[SUP] 1 [/SUP], Yunhua Gao[SUP] 1 [/SUP], Xiaoting Qiao[SUP] 1 [/SUP], Jie Xie[SUP] 1 [/SUP], Yongzhuo Zhang[SUP] 1 [/SUP], Di Wang[SUP] 1 [/SUP], Lianhua Dong[SUP] 2 [/SUP]
Affiliations
- PMID: 35941317
- DOI: 10.1007/s00216-022-04238-6
Abstract
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.
Keywords: Digital PCR; IDMS; Reverse transcription; SARS-CoV-2.