key: cord-0827019-tlb7056m 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 title: Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnostic date: 2020-10-29 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2020.10.079 sha: a1a6cc1e8ada302dcf269e1e4ef8751046ee3e0e doc_id: 827019 cord_uid: tlb7056m The 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 diagnostic routine, and propose alternatives to reduce dimerization events. Late unspecific amplifications were visualized in 56.4% and 57.1% of negative samples and no-template control, respectively, but not in positive samples and positive control. In silico analysis and gel electrophoresis confirm 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 that require repetition of tests, which increases the costs and generates delays in results or even unnecessary requests for new samples. 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According to CDC protocol (CDC, 2020), the single amplification of any of the specific virus targets (N1/N2) results in "inconclusive result", and the recommendation is to repeat the analysis. As observed in routine assays, late N2 amplifications frequently appears in negative samples turning it "inconclusive". Thus, we decided to study the late unspecific amplifications in negative samples when using the CDC 2019-nCoV RT-qPCR protocol and to propose alternatives to reduce these events. RNA was extracted from samples collected from nasopharynx and oropharynx using Material 1) . A partial sequence homology between N2 probe-probe (selfdimer, Figure 1E ), N2 forward primer-probe and N2 reverse primer-probe (hetero-dimer, Figure 1F and G, respectively) of more than 3 base pair were observed, with Delta G values of -13.09, -8.98, and -9.89 Kcal/mol, respectively. Moreover, there is N2 forward primer-probe homology at the 3' end of the sequence (Fig. 1F ). Hairpin loops in N2 primers and probe showed Delta G values close to zero. Interestingly, we observed fragment size of less than 50bp in negative samples ( Figure 1C , lanes 3, 4, and 5). As the expected fragment of 2019-nCoV_N Positive Control and positive samples is 72bp ( Figure 1C , lanes 2 and 6, 7, respectively), PCR products <50bp confirm the dimerization of primers and/or probes. In order to minimize the dimer formation in N2 reactions, we tested different RT-qPCR conditions, including concentration of primers (133, 213, 266, 320, and 400nM), probe (33, 54, 67.5, 81, and 100nM), and MgSO4 (3, 4, 5, 6, and 6.5mM appear to be exclusive to nucleocapsid targets. Unspecific signals at late cycles in the envelope protein gene (E target) assay using the Charité protocol (Konrad et al., 2020) and mismatch of primers sequences (Pillonel et al., 2020) were recently reported. The scientific community is discussing the technical limitations in the SARS-CoV-2 RT-qPCR protocols (Marx, 2020) and its optimization is still underway. It is known that primers-probe set are the pivotal component of a qPCR assay (Bustin and Huggett, 2017) and if the dimerization occurs in a staggered manner, some extension can occur and become more abundant as cycling progresses. The in silico analysis showed that probe-probe self-dimerfor instance -had potential to bind at the 5'and 3' ends, requiring high amount of energy to fully break a secondary DNA structure (Delta G=-13.09Kcal/mol). Despite this, the optimization presented here has drastically reduced the dimerization events. The main concern with primer-dimers formation is that they may cause false-positive results. In our experience, 56.4% of the not detected reactions (negative samples) showed the late unspecific amplification. The strict adjustment in the RT-qPCR conditions carried out in the present study was decisive for the optimization of the reaction and reduction of the occurrence of late unspecific amplifications (11.5%). In addition, the single amplification of any of the specific virus targets (N1/N2)even though it is an unspecific J o u r n a l P r e -p r o o f amplification -results in "inconclusive result" that require repetition of tests increasing the costs and generating delays in results or even unnecessary requests for new samples. There was no decrease in the efficiency of reactions in positive control and positive samples, even though the dimerization events on N2 primers-probe set is suggested. This may be due to preferential annealing of the primers and probe to cDNA template of positive samples, which occur in earlier cycles of PCR (cycles 10-30, depending on the amount of viral genetic material). Although the detection of SARS-CoV-2 in positive samples seems not to be affected by unspecific signals, it has great importance in negative samples assessment, leading to inconclusive results. Finally, we recommend RT-qPCR users to adjust primers-probe and magnesium concentration, RT step duration, and thermal cycle temperature, independently of the Master mix kit used, to minimize dimer formation and to avoid extensive test repetition and the waste of resources. The authors declare that they have no competing interests This work was supported by Universidade Federal de Juiz de Fora (UFJF). qPCR primer design revisited Researchers develop first diagnostic test for novel coronavirus in China Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Rapid establishment of laboratory diagnostics for the novel coronavirus SARS-CoV-2 in Bavaria Coronavirus jolts labs to warp speed Pan American Health Organization, Laboratory Guidelines for Detection and Diagnosis of the Novel Coronavirus (2019-nCoV) Infection detection by real-time RT-PCR Triplex Real-Time RT-PCR for Severe Acute Respiratory Syndrome Coronavirus 2 Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19) We thank M.I.N. Di Azevedo for editing the agarose gel images.