Validation of the easyscreen flavivirus dengue alphavirus detection kit based on 3base amplification technology and its application to the 2016/17 Vanuatu dengue outbreak.

Validation of the easyscreen flavivirus dengue alphavirus detection kit based on 3base amplification technology and its application to the 2016/17 Vanuatu dengue outbreak.

Blog Article

BACKGROUND:The family flaviviridae and alphaviridae contain a diverse group of pathogens that cause significant morbidity and mortality worldwide.Diagnosis of the virus responsible for disease is essential to ensure patients receive appropriate clinical management.Very few real-time RT-PCR based assays are able to detect the presence of all members of these families using a single primer and probe set.

We have developed a novel chemistry, 3base, which simplifies the viral nucleic acids allowing the design of RT-PCR assays capable of pan-family identification.METHODOLOGY/PRINCIPAL FINDING:Synthetic constructs, viral nucleic acids, intact viral particles and characterised reference materials were used to determine the specificity and sensitivity of the assays.Synthetic constructs demonstrated the sensitivities of the pan-flavivirus detection component were in the range of 13 copies per PCR.

The pan-alphavirus assay had LIVING MUSTARD a sensitivity range of 10-25 copies per reaction depending on the viral strain.Lower limit of detection studies using whole virus particles demonstrated that sensitivity for assays was in the range of 1-2 copies per reaction.No cross reactivity was observed with a number of commonly encountered viral strains.

Proficiency panels showed 100% concordance with the expected results and the assays performed as well as, if not better than, other assays used in laboratories worldwide.After initial assay validation the pan-viral assays were then tested during the 2016-2017 Vanuatu dengue-2 outbreak.Positive results were detected in 116 positives from a total of 187 suspected dengue samples.

CONCLUSIONS/SIGNIFICANCE:The pan-viral screening assays described here utilise a novel 3base technology and are PLANT STEROLS shown to provide a sensitive and specific method to screen and thereafter speciate flavi- and/or alpha- viruses in clinical samples.The assays performed well in an outbreak situation and can be used to detect positive clinical samples containing any flavivirus or alphavirus in approximately 3 hours 30 minutes.