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Real-time molecular techniques have become the reference methods for direct diagnosis of pathogens. The reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. We describe here a process of preparation of oligonucleotide primers and hydrolysis probe in a single tube at predefined optimized concentrations that are stabilized via lyophilization (Lyoph-P&P). Lyoph-P&P was compared versus the classic protocol using extemporaneously prepared liquid reagents using (i) sensitivity study, (ii) long-term stability at 4 °C, and (iii) long-term stability at 37 °C mimicking transportation without cold chain. Two previously published molecular assays were selected for this study. They target two emerging viruses that are listed on the blueprint of the WHO as to be considered for preparedness and response actions: chikungunya virus (CHIKV) and Rift Valley fever phlebovirus (RVFV). Results of our study demonstrate that (i) Lyoph-P&P is stable for at least 4 days at 37 °C supporting shipping without the need of cold chain, (ii) Lyoph-P&P rehydrated solution is stable at +4 °C for at least two weeks, (iii) sensitivity observed with Lyoph-P&P is at least equal to, often better than, that observed with liquid formulation, (iv) validation of results observed with low-copy specimens is rendered easier by higher fluorescence level. In conclusion, Lyoph-P&P holds several advantages over extemporaneously preparer liquid formulation that merit to be considered when a novel real-time molecular assay is implemented in a laboratory in charge of routine diagnostic activity.

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Unité des Virus Emergents (UVE: Aix Marseille Univ, IRD 190, INSERM 1207, IHU Méditerranée Infection), 13005 Marseille, France.