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

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 Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome.

Preparedness and response actions to mitigate Ebola virus disease (EVD) outbreaks rely on rapid diagnosis to be implemented locally to sort suspect patients attending health centers. Our aim was (i) to develop and evaluate an RT-qPCR assay combining primers and probes derived from two reference assays targeting different genomic regions; (ii) to study whether sensitivity and specificity of this dual-target assay were at least equal or better to the parental assays; (iii) to implement this dual-target assay onto the Cepheid GeneXpert open cartridge as a proof of principle for technological transfer aiming at bedsite testing locally. To do so, three home-made published RT-qPCR assays were selected to be compared with the RealStar® Filovirus Screen RT-PCR kit 1.0 (Altona Diagnostics, Hamburg, Germany), a technique that was largely deployed during the 2014-2015 West African EVD outbreak. Primers and probes sequences of the custom-made assays were analyzed in silico against a multiple sequence alignment, including >250 complete sequences corresponding to strains that have caused EVD epidemics in the past. Genomic RNA purified from the Mekambo strain of Zaire ebolavirus (EBOV) was used to study the sensitivity of the five methods. Based on these results, two in-house methods were selected and adapted to design the dual-target assay, which performances were compared to those of the parental assays using a synthetic RNA control. The dual-target assay showed better sensitivity and limit of detection (LoD95 at 0.4 copies/µL) than the parental methods (1.7 and 2.2 copies/µL). Ultimately, the dual-target assay was transferred onto the GeneXpert Flex-03 open cartridge, demonstrating a LoD95 at 0.75 copies/µL. Together these results indicate that EBOV dual-target assay has the potential to be used during EVD outbreak in the laboratory having performed molecular testing during the recent outbreaks.

Development and Evaluation of a Duo Chikungunya Virus Real-Time RT-PCR Assay Targeting Two Regions within the Genome.

Chikungunya virus (CHIKV) re-emerged as a globalized health threat fifteen years ago. There are dozens of RT-PCR assays published. An inventory of the latter was made, and after in silico analysis, two assays were selected for their ability to detect strains belonging to the five CHIKV genetic lineages. They were combined in order to provide a robust assay not affected by genetic point mutations and the resulting Duo CHIKV real-time RT-PCR assay was compared to the two parental single-plex tests against five strains belonging to the five genetic lineages. The Duo CHIKV assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. Dual-target assays are better suited for viruses having the propensity to evolve into new variants via point mutations or major sequence deletions/insertions. Here, we demonstrated that combining two single systems into a dual-target assay did not impair sensitivity and specificity, and proved a potent diagnostic tool to face a potential emergence of CHIKV variants by newly evolving mutations.