Lyophilized Matrix Containing Ready-to-Use Primers and Probe Solution for Standardization of Real-Time PCR and RT-qPCR Diagnostics in Virology

Abstract

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.

Development and Evaluation of a duo SARS-CoV-2 RT-qPCR Assay Combining Two Assays Approved by the World Health Organization Targeting the Envelope and the RNA-Dependant RNA Polymerase (RdRp) Coding Regions

“…These preliminary results suggest that clinical samples can be tested using a unique RT-qPCR reaction; this might be of utmost importance to overcome the problems due to molecular biology reagent shortage, and to allow larger testing capacity for clinical microbiology laboratories involved in the COVID-19 response actions. The primers and probes presented in this study are publicly available for academics and industrials in the European Virus Archive catalog in the lyophilized format previously described [ 18 ] and can beneficiate from the free of charge access through the Trans National Access policy of the EVA-GLOBAL project [ 19 , 20 , 21 ].…”

Evaluation of a Trio Toscana Virus Real-Time RT-PCR Assay Targeting Three Genomic Regions within Nucleoprotein Gene

“…The Trio TOSV reagents, in the form of Lyoph-P&P vials containing 24 reactions each (as previously described [25]), were sent to our Spanish partner who performed comparative analysis of Trio TOSV versus the assay routinely used in its laboratory for TOSV RNA detection [23]. The comparison was performed using two different Master mixes: (i) qScript™ XLT One-Step RT-qPCR (Quanta Bio, VWR International Eurolab S.L., Llinars del Vallès, Spain) and (ii) RealTime ready RNA Virus Master (Roche Diagnostics, Barcelona, Spain).…”

Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics

“…Several publications on freeze-dried PCR mixes have been reported during the past 20 years ( Table S1 ) [ [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] ]. In previous studies, electrophoresis has frequently been used to evaluate the activities of freeze-dried PCR reagents [ 9 , 10 , 12 ].…”