Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis. (January 2021)

• Record Type: Journal Article
• Authors: Jaeger, Lauren Hubert; Nascimento, Thiago César; Rocha, Fabíola Dutra; Vilela, Fernanda Maria Pinto; Duque, Ana Paula do Nascimento; Silva, Lívia Mara; Riani, Lorena Rodrigues; Moreira, João Paulo; Chagas, Jéssica Mara de Assis; Pereira, Thamiris Vilela; Perches, Carmen Gomide Pinto; Watanabe, Aripuana Sakurada Aranha; Viccini, Lyderson Facio; Silvério, Marcelo Silva; Corrêa, José Otávio do Amaral; Pereira-Junior, Olavo dos Santos; Pittella, Frederico
• Is Part Of: International journal of infectious diseases. Volume 102(2021)
• Page Start: 437
• Page End: 439
• Publication Date: 2021-01
• Subjects: Coronavirus -- Diagnostic techniques and procedures -- COVID-19 -- RT-qPCR; Communicable diseases -- Periodicals; Communicable Diseases -- Periodicals; Communicable diseases; Periodicals; Electronic journals; 616.9
• DOI: 10.1016/j.ijid.2020.10.079
• Languages: English
• ISSNs: 1201-9712

Abstract

Highlights: Unspecific amplifications were found in 56.4% (495 reactions) of samples that were negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In silico analysis of N2 primers–probe and gel electrophoresis showed dimer formation. Optimization of RT-qPCR conditions reduced the dimerization events. Conditions must be adjusted to avoid extensive test repetition and the waste of resources. Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and quickly spread around the world, forcing global health authorities to develop protocols for its diagnosis. Here we report dimer formation in the N2 primers–probe set (CDC 2019-nCoV Real-Time RT-PCR) used in the diagnostic routine, and propose alternatives to reduce dimerization events. Late unspecific amplifications were visualized in 56.4% of negative samples and 57.1% of no-template control, but not in positive samples or positive control. In silico analysis and gel electrophoresis confirmed the dimer formation. The RT-qPCR parameters were optimized and the late unspecific amplifications decreased to 11.5% in negative samples and no-template control. The adjustment of PCR parameters was essential to reduce the risk of false-positives results and to avoid inclusive results requiring repeat testing, which increases the costs and generates delays in results or even unnecessary requests for new samples.