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Thi Nguyen, Ngoc Anh; Thi Bui, Hoai; Thi-Huong Pham, Quynh; Thi Thao Hoang, Ly; Xuan Ta, Hung; Heikkinen, Timo; Van Le, Duyet; Dinh Van, Trang; Quoc Ngo, Nam; Thi Hong Huynh, Phuong; Thi Huyen Tran, Trang; Quoc Phan, Hoan; Van Hoang, Luong; van Doorn, H.Rogier; Thi Ngoc Nguyen, Diep; Thi Nguyen, Tam; Sy Vo, Nam; Viet Vo, Cuong; Khac Trinh, Sau; The Pham, Tai; Duc Le, Quang; Van Le, Phan; Thai Nguyen, Son; Thi Tran, Loan; Dinh Vu, Toan; Vu Nguyen, Quynh Anh; Thi Trieu, Nguyet; Thi Le, Thuy; Dinh Nguyen, Ung; Steman, Jakob; Huu Ho, Tho
Clinica chimica acta, 06/2022, Volume: 531Journal Article
Display omitted •A semi-nested, heptaplex RT-PCR assay for SARS-CoV-2 detection has been developed.•The complex melting spectrum was interpreted by an artificial intelligence algorithm.•The developed assay enables 96-sample pooled testing for increase of testing capacity.•About 8,000 pre-amplified samples could be screened in one realtime PCR run. Asymptomatic transmission was found to be the Achilles’ heel of the symptom-based screening strategy, necessitating the implementation of mass testing to efficiently contain the transmission of COVID-19 pandemic. However, the global shortage of molecular reagents and the low throughput of available realtime PCR facilities were major limiting factors. A novel semi-nested and heptaplex (7-plex) RT-PCR assay with melting analysis for detection of SARS-CoV-2 RNA has been established for either individual testing or 96-sample pooled testing. The complex melting spectrum collected from the heptaplex RT-PCR amplicons was interpreted with the support of an artificial intelligence algorithm for the detection of SARS-CoV-2 RNA. The analytical and clinical performance of the semi-nested RT-PCR assay was evaluated using RNAs synthesized in-vitro and those isolated from nasopharyngeal samples. The LOD of the assay for individual testing was estimated to be 7.2 copies/reaction. Clinical performance evaluation indicated a sensitivity of 100% (95% CI: 97.83–100) and a specificity of 99.87% (95% CI: 99.55–99.98). More importantly, the assay supports a breakthrough sample pooling method, which makes possible parallel screening of up to 96 samples in one real-time PCR well without loss of sensitivity. As a result, up to 8,820 individual pre-amplified samples could be screened for SARS-CoV-2 within each 96-well plate of realtime PCR using the pooled testing procedure. The novel semi-nested RT-PCR assay provides a solution for highly multiplex (7-plex) detection of SARS-CoV-2 and enables 96-sample pooled detection for increase of testing capacity. .
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