Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis
Lauren Hubert Jaeger, Thiago César Nascimento, Fabíola D. Rocha, Fernanda Maria Pinto Vilela, Ana Paula do Nascimento Duque, Lívia Mara Silva, Lorena Rodrigues Riani, João Moreira, Jéssica Mara de Assis Chagas, Thamiris Vilela Pereira, Carmen Gomide Pinto Perches, Aripuanã Sakurada Aranha Watanabe, Lyderson Fácio Viccini, Marcelo Silva Silvério, José Otávio do Amaral Corrêa, Olavo dos Santos Pereira-Júnior, Frederico Pittella
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.