Universal detection of all LASV lineages by PANDAA
Detection of ten LASV lineage reference strains at 50 RNA copies / reaction. LCMV—one of the most closely-related arenaviruses—does not cross the Ct cut-off of 30 cycles when used at 100-fold excess of the LASV RNA copies (5,000 copies / reaction).
Highly sensitive detection down to 10 copies / reaction.
The limit of detection is shown for LASV reference strain Josiah. Serial dilutions were performed from 5×107 RNA copies to 2 RNA copies per reaction (blue). A Ct cut-off of 30 cycles was set to make a positive call. Although 2 copies / RNA are detected (orange), they are above the Ct cut-off. The human genomic DNA negative control (red) can be differentiated clearly from the 10 and 2 copies / reaction. Panel (A) shows the amplification curves with the threshold and Ct cut-off; Panel (B) shows the linear relationship between input copy number and Ct.
PANDAA is highly specific for LASV.
PANDAA specificity was evaluated using a panel of 16 pathogens covering related arenavirus, other VHFs, and pathogens that may present in patients in West Africa. Full length isolated RNA / DNA was obtained from BEI Resources and used at a minimum of 5×10^5 copies / reaction, a 50,000-fold excess of the assay limit of detection of 10 RNA copies / reaction. None of the specificity panel members crossed the Ct cut-off of 30 cycles, and 10 copies / reaction of the LASV strain Nig08-A37 was clearly distinguishable. Data represent mean Ct value and 95% CI.
PANDAA significantly improves sensitivity and rescues LASV detection compared to conventional qPCR.
PANDAA’s adaptative PCR approach utilizes primers and probes that are capable of tolerating and adapting probe-binding site polymorphisms. As proof-of-concept, PANDAA was compared to conventional qPCR, and was shown to increase sensitivity by an average of 50-fold for the most divergent LASV strains and rescued the detection of isolates that gave false negative. It is clear that, regardless of the LASV strain used, PANDAA returns comparable Ct values. However, with conventional qPCR, the sensitivity of the LASV diagnostic is highly variable, and for two LASV strains results in a false negative result. Data shown use 10^4 copies / reaction.