tetano
Editor, Senior Moderator
PLoS One
. 2021 Jun 4;16(6):e0252789.
doi: 10.1371/journal.pone.0252789. eCollection 2021.
Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection
Yukiko Nakura[SUP] 1 [/SUP], Heng Ning Wu[SUP] 1 [/SUP], Yuya Okamoto[SUP] 2 [/SUP], Muneyuki Takeuchi[SUP] 3 [/SUP], Koichiro Suzuki[SUP] 4 [/SUP], Yoshitaka Tamura[SUP] 5 [/SUP], Yuichiro Oba[SUP] 6 [/SUP], Fumiko Nishiumi[SUP] 1 [/SUP], Nobuaki Hatori[SUP] 4 [/SUP], Shinsuke Fujiwara[SUP] 7 [/SUP], Kiyoshi Yasukawa[SUP] 8 [/SUP], Shinobu Ida[SUP] 2 [/SUP], Itaru Yanagihara[SUP] 1 2 [/SUP]
Affiliations
Abstract
The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method's sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.
. 2021 Jun 4;16(6):e0252789.
doi: 10.1371/journal.pone.0252789. eCollection 2021.
Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection
Yukiko Nakura[SUP] 1 [/SUP], Heng Ning Wu[SUP] 1 [/SUP], Yuya Okamoto[SUP] 2 [/SUP], Muneyuki Takeuchi[SUP] 3 [/SUP], Koichiro Suzuki[SUP] 4 [/SUP], Yoshitaka Tamura[SUP] 5 [/SUP], Yuichiro Oba[SUP] 6 [/SUP], Fumiko Nishiumi[SUP] 1 [/SUP], Nobuaki Hatori[SUP] 4 [/SUP], Shinsuke Fujiwara[SUP] 7 [/SUP], Kiyoshi Yasukawa[SUP] 8 [/SUP], Shinobu Ida[SUP] 2 [/SUP], Itaru Yanagihara[SUP] 1 2 [/SUP]
Affiliations
- PMID: 34086827
- DOI: 10.1371/journal.pone.0252789
Abstract
The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method's sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.