tetano
Editor, Senior Moderator
Microbiol Spectr
. 2024 Jun 25:e0429223.
doi: 10.1128/spectrum.04292-23. Online ahead of print. Sensitivity analysis of RT-qPCR and RT-ddPCR for SARS-CoV-2 detection with mutations on N1 and E primer-probe region
Shiyou Liu[SUP] #[/SUP][SUP] 1 2 [/SUP], Xiaoru Chai[SUP] #[/SUP][SUP] 2 [/SUP], Chao Liu[SUP] 3 [/SUP], Jiaxuan Bai[SUP] 2 [/SUP], Juntao Meng[SUP] 2 [/SUP], Hong Tian[SUP] 2 [/SUP], Xu Han[SUP] 1 [/SUP], Guangyue Han[SUP] 1 [/SUP], Qi Li[SUP] 1 2 [/SUP], Xiangdong Xu[SUP] 2 4 [/SUP]
Affiliations
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus that undergoes rapid mutation. Based on viral whole genome sequencing analysis in Hebei Province, China, we identified several essential single nucleotide variants (SNVs) on primer-probe regions accumulating within some Omicron variants' genomes. In this study, we focused on three SNVs, C28290T, T28297C, and C28311T emerging on 2019-nCoV-N1 (CDC-N1) primer-probe regions, recommended by CDC in 2020, and two SNVs, C26270T, A26275G emerging on E (Charité-E) primer-probe regions recommended by Charité, Germany. Our findings revealed that the presence of one or two SNVs in the primer or probe region affected the sensitivity of reverse transcription-quantitative polymerase chain reaction and droplet digital PCR to varying extents. This discovery underscores the importance of continuously monitoring the whole genome sequences of SARS-CoV-2 variants, especially the primer-probe targeting regions, and correspondingly updating commercial test kits or recommended primer-probe sequence sets.
Importance: The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in a growing number of mutations in its genome, presenting new challenges for the diagnosis of SARS-CoV-2 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and droplet digital PCR (RT-ddPCR) methods. There is an urgent need to develop refined methods for modifying primers and probes to improve the detection of these emerging variants. In this study, our focus was on the SNVs that have emerged in the CDC-N1 and Charité-E primer-probe regions. Our research has confirmed that the presence of these SNVs in the primer or probe region can significantly affect the results of coronavirus disease 2019 tests. we have developed and validated a modified detection method that can provide higher sensitivity and specificity. This study emphasizes the importance of refining the primer-probe sets to ensure the diagnostic accuracy of RT-qPCR and RT-ddPCR detection.
Keywords: RT-ddPCR; RT-qPCR; SARS-CoV-2; mutation; primer-probe region.
. 2024 Jun 25:e0429223.
doi: 10.1128/spectrum.04292-23. Online ahead of print. Sensitivity analysis of RT-qPCR and RT-ddPCR for SARS-CoV-2 detection with mutations on N1 and E primer-probe region
Shiyou Liu[SUP] #[/SUP][SUP] 1 2 [/SUP], Xiaoru Chai[SUP] #[/SUP][SUP] 2 [/SUP], Chao Liu[SUP] 3 [/SUP], Jiaxuan Bai[SUP] 2 [/SUP], Juntao Meng[SUP] 2 [/SUP], Hong Tian[SUP] 2 [/SUP], Xu Han[SUP] 1 [/SUP], Guangyue Han[SUP] 1 [/SUP], Qi Li[SUP] 1 2 [/SUP], Xiangdong Xu[SUP] 2 4 [/SUP]
Affiliations
- PMID: 38916349
- DOI: 10.1128/spectrum.04292-23
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus that undergoes rapid mutation. Based on viral whole genome sequencing analysis in Hebei Province, China, we identified several essential single nucleotide variants (SNVs) on primer-probe regions accumulating within some Omicron variants' genomes. In this study, we focused on three SNVs, C28290T, T28297C, and C28311T emerging on 2019-nCoV-N1 (CDC-N1) primer-probe regions, recommended by CDC in 2020, and two SNVs, C26270T, A26275G emerging on E (Charité-E) primer-probe regions recommended by Charité, Germany. Our findings revealed that the presence of one or two SNVs in the primer or probe region affected the sensitivity of reverse transcription-quantitative polymerase chain reaction and droplet digital PCR to varying extents. This discovery underscores the importance of continuously monitoring the whole genome sequences of SARS-CoV-2 variants, especially the primer-probe targeting regions, and correspondingly updating commercial test kits or recommended primer-probe sequence sets.
Importance: The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in a growing number of mutations in its genome, presenting new challenges for the diagnosis of SARS-CoV-2 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and droplet digital PCR (RT-ddPCR) methods. There is an urgent need to develop refined methods for modifying primers and probes to improve the detection of these emerging variants. In this study, our focus was on the SNVs that have emerged in the CDC-N1 and Charité-E primer-probe regions. Our research has confirmed that the presence of these SNVs in the primer or probe region can significantly affect the results of coronavirus disease 2019 tests. we have developed and validated a modified detection method that can provide higher sensitivity and specificity. This study emphasizes the importance of refining the primer-probe sets to ensure the diagnostic accuracy of RT-qPCR and RT-ddPCR detection.
Keywords: RT-ddPCR; RT-qPCR; SARS-CoV-2; mutation; primer-probe region.