tetano
Editor, Senior Moderator
Sci Rep
. 2024 Sep 4;14(1):20590.
doi: 10.1038/s41598-024-71705-8. ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay
Ekasit Kowitdamrong[SUP] 1 2 [/SUP], Sasiprapa Anoma[SUP] 3 4 [/SUP], Thitiya Loykaew[SUP] 5 [/SUP], Pokrath Hansasuta[SUP] 3 [/SUP], Parvapan Bhattarakosol[SUP] 3 4 [/SUP]
Affiliations
Real-time reverse transcription polymerase chain reaction (RT-PCR), a standard method recommended for the diagnosis of coronavirus disease 2019 (COVID-19) requires 2-4 h to get the result. Although antigen test kit (ATK) is used for COVID-19 screening within 15-30 min, the drawback is its limited sensitivity. Hence, a rapid one-step quadruplex real-time RT-PCR assay: termed ƩS COVID-19 targeting ORF1ab, ORF3a, and N genes of SARS-CoV-2; and Avocado sunblotch viroid (ASBVd) as an internal control was developed. Based on strategies including designing high melting temperature primers with short amplicons, applying a fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing/extension temperatures; the assay could be accomplished within 25 min. The limit of detection of ORF1ab, ORF3a, and N genes were 1.835, 1.310, and 1 copy/reaction, respectively. Validation was performed in 205 combined nasopharyngeal and oropharyngeal swabs. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.8%, 100%, 100%, and 97.1%, respectively with 96.7% accuracy. Cohen's Kappa was 0.93. The newly developed rapid real-time RT-PCR assay was highly sensitive, specific, and fast, making it suitable for use as an alternative method to support laboratory diagnosis of COVID-19 in outpatient and emergency departments.
Keywords: COVID-19; High throughput; Rapid; Real time RT-PCR; SARS-CoV-2.
. 2024 Sep 4;14(1):20590.
doi: 10.1038/s41598-024-71705-8. ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay
Ekasit Kowitdamrong[SUP] 1 2 [/SUP], Sasiprapa Anoma[SUP] 3 4 [/SUP], Thitiya Loykaew[SUP] 5 [/SUP], Pokrath Hansasuta[SUP] 3 [/SUP], Parvapan Bhattarakosol[SUP] 3 4 [/SUP]
Affiliations
- PMID: 39232060
- DOI: 10.1038/s41598-024-71705-8
Real-time reverse transcription polymerase chain reaction (RT-PCR), a standard method recommended for the diagnosis of coronavirus disease 2019 (COVID-19) requires 2-4 h to get the result. Although antigen test kit (ATK) is used for COVID-19 screening within 15-30 min, the drawback is its limited sensitivity. Hence, a rapid one-step quadruplex real-time RT-PCR assay: termed ƩS COVID-19 targeting ORF1ab, ORF3a, and N genes of SARS-CoV-2; and Avocado sunblotch viroid (ASBVd) as an internal control was developed. Based on strategies including designing high melting temperature primers with short amplicons, applying a fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing/extension temperatures; the assay could be accomplished within 25 min. The limit of detection of ORF1ab, ORF3a, and N genes were 1.835, 1.310, and 1 copy/reaction, respectively. Validation was performed in 205 combined nasopharyngeal and oropharyngeal swabs. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.8%, 100%, 100%, and 97.1%, respectively with 96.7% accuracy. Cohen's Kappa was 0.93. The newly developed rapid real-time RT-PCR assay was highly sensitive, specific, and fast, making it suitable for use as an alternative method to support laboratory diagnosis of COVID-19 in outpatient and emergency departments.
Keywords: COVID-19; High throughput; Rapid; Real time RT-PCR; SARS-CoV-2.