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2023 |
Rapid Detection of SARS-CoV-2 RNA Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) with Lateral Flow for N-Protein Gene and Variant-Specific Deletion-Insertion Mutation in S-Protein Gene |
Jose L Malaga, Monica J Pajuelo, Michiko Okamoto, Emmanuel Kagning Tsinda, Kanako Otani, Pablo Tsukayama, Lucero Mascaro, Diego Cuicapuza, Masamichi Katsumi, Kazuhisa Kawamura, Hidekazu Nishimura, Akie Sakagami, Yo Ueki, Suguru Omiya, Satoshi Okamoto, Asami Nakayama, Shin-Ichi Fujimaki, Chuyao Yu, Sikandar Azam, Eiichi Kodama, Clyde Dapat, Hitoshi Oshitani, Mayuko Saito |
Viruses |
37376555 |
10.3390/v15061254 |
Rapid Detection of SARS-CoV-2 RNA Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) with Lateral Flow for N-Protein Gene and Variant-Specific Deletion-Insertion Mutation in S-Protein Gene
Author(s):
Jose L Malaga, Monica J Pajuelo, Michiko Okamoto, Emmanuel Kagning Tsinda, Kanako Otani, Pablo Tsukayama, Lucero Mascaro, Diego Cuicapuza, Masamichi Katsumi, Kazuhisa Kawamura, Hidekazu Nishimura, Akie Sakagami, Yo Ueki, Suguru Omiya, Satoshi Okamoto, Asami Nakayama, Shin-Ichi Fujimaki, Chuyao Yu, Sikandar Azam, Eiichi Kodama, Clyde Dapat, Hitoshi Oshitani, Mayuko Saito
Journal:
Viruses
Year:
2023
Abstract:
Rapid molecular testing for severe acute respiratory coronavirus 2 (SARS-CoV-2) variants may contribute to the development of public health measures, particularly in resource-limited areas. Reverse transcription recombinase polymerase amplification using a lateral flow assay (RT-RPA-LF) allows rapid RNA detection without thermal cyclers. In this study, we developed two assays to detect SARS-CoV-2 nucleocapsid (N) gene and Omicron BA.1 spike (S) gene-specific deletion-insertion mutations (del211/ins214). Both tests had a detection limit of 10 copies/µL in vitro and the detection time was approximately 35 min from incubation to detection. The sensitivities of SARS-CoV-2 (N) RT-RPA-LF by viral load categories were 100% for clinical samples with high (>9015.7 copies/µL, cycle quantification (Cq): < 25) and moderate (385.5-9015.7 copies/µL, Cq: 25-29.9) viral load, 83.3% for low (16.5-385.5 copies/µL, Cq: 30-34.9), and 14.3% for very low (<16.5 copies/µL, Cq: 35-40). The sensitivities of the Omicron BA.1 (S) RT-RPA-LF were 94.9%, 78%, 23.8%, and 0%, respectively, and the specificity against non-BA.1 SARS-CoV-2-positive samples was 96%. The assays seemed more sensitive than rapid antigen detection in moderate viral load samples. Although implementation in resource-limited settings requires additional improvements, deletion-insertion mutations were successfully detected by the RT-RPA-LF technique.
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