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

• Published in: Viruses Vol. 15; no. 6; p. 1254
• Main Authors: Malaga, Jose L, Pajuelo, Monica J, Okamoto, Michiko, Tsinda, Emmanuel Kagning, Otani, Kanako, Tsukayama, Pablo, Mascaro, Lucero, Cuicapuza, Diego, Katsumi, Masamichi, Kawamura, Kazuhisa, Nishimura, Hidekazu, Sakagami, Akie, Ueki, Yo, Omiya, Suguru, Okamoto, Satoshi, Nakayama, Asami, Fujimaki, Shin-Ichi, Yu, Chuyao, Azam, Sikandar, Kodama, Eiichi, Dapat, Clyde, Oshitani, Hitoshi, Saito, Mayuko
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 26.05.2023; MDPI
• Subjects: antigen detection; Coronaviruses; COVID-19; COVID-19 - diagnosis; COVID-19 - genetics; deletion–insertion mutation; Design; detection limit; Disease transmission; Enzymes; Gene deletion; Gene flow; genes; Genomes; Humans; Insertion; Molecular diagnostic techniques; Mutagenesis, Insertional; Mutation; Nucleic Acid Amplification Techniques - methods; nucleocapsid; Nucleocapsids; Nucleotidyltransferases - genetics; Proteins; Public health; rapid methods; Recombinase; recombinase polymerase amplification; recombinase polymerase amplification (RPA); Recombinases - genetics; Reverse Transcription; RNA; RNA, Viral - genetics; SARS-CoV-2; SARS-CoV-2 - genetics; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; Technical Note; variant of concern (VOC); viral load; Peru; United States > US; Japan; COVID-19; recombinase polymerase amplification (RPA); SARS-CoV-2; deletion–insertion mutation; variant of concern (VOC)
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v15061254

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.