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.
Highlights
The detection of the genome of pathogens has become the gold standard technique for direct diagnosis because of excellent sensitivity and specificity, and due to its capacity to provide a result within hours [1,2]
We describe here a process of preparation of oligonucleotide primers and hydrolysis probe in a single tube at predefined optimized concentrations (P&P for Primers and Probe(s)) that are stabilized via lyophilization (Lyoph-Primers & Probe mix (P&P))
Selected assays target two emerging viruses that are listed on the blueprint of the WHO as to be considered for preparedness and response actions [5]: chikungunya virus (CHIKV), a single-stranded positive-sense RNA alphavirus, and Rift Valley fever phlebovirus (RVFV), a tri-segmented, single-stranded negative-sense RNA phlebovirus
Summary
The detection of the genome of pathogens has become the gold standard technique for direct diagnosis because of excellent sensitivity and specificity, and due to its capacity to provide a result within hours [1,2]. Failure of one of the mix components is detected when the positive control does not provide adequate results, such a situation has an important impact on the laboratory throughput due to delayed results, reordering reagents, increased laboratory costs, increasing technical workload, and feeling insecure concerning the capacity of biologists to provide results and of clinicians to obtain results timely Whether this can appear as a minor problem for laboratories using few in-house assays, it can rapidly become hectic when a larger number of in-house assays are used for routine diagnostic purpose. There are several causes for failures linked to primers and/or probes such as light exposure that deteriorates fluorescence of the probe, repeated freeze-thaw cycles resulting in DNA degradation, mistakes in final concentrations, or pipetting errors when the reaction mix is prepared [4] Such problems have been at least partially solved in commercial kits through serial aliquoting and lyophilization or ambient-temperature stable reagents. Selected assays target two emerging viruses that are listed on the blueprint of the WHO as to be considered for preparedness and response actions [5]: chikungunya virus (CHIKV), a single-stranded positive-sense RNA alphavirus, and Rift Valley fever phlebovirus (RVFV), a tri-segmented, single-stranded negative-sense RNA phlebovirus
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