Fig 1.
YFV RNA extraction from FTA punches lowers the sensitivity of YFV qRT-PCR assays based on volume, not technique.
RNA from the vaccine strain of YFV, 17D-204, was extracted using the manufacturers protocol from 140 μL of YFV diluted in PBS, from a single FTA punch, from 10 μL of YFV diluted in PBS, and from two FTA punches. The dotted line at Ct value 37 indicates the cut off for YFV RNA positivity and is based on the cut-off used in the molecular diagnosis of YFV in the field. Dilutions to make the standard curve were performed in PBS. Points represent the average of two experiments and were fit using a linear regression.
Fig 2.
YFV spiked into serum can be extracted from FTA cards but with lower sensitivity than traditional methods.
RNA from the vaccine strain of YFV, 17D-204, was extracted using the manufacturers protocol from 140 μL of YFV diluted in serum, from a single FTA punch, and from two FTA punches. The dotted line at Ct value 37 indicates the cut-off for YFV RNA positivity and is based on cutoffs used in the molecular diagnosis of YFV in the field. Dilution to make the standard curve were performed in human, flavivirus negative serum. Points represent the average of two experiments and were fit using a linear regression.
Fig 3.
FTA card stabilization of YFV RNA decrease at high temperature.
The vaccine strain of YFV, 17D-204, was diluted in PBS, spotted onto punches from FTA cards and placed at RT (~25°C) or 37°C for two weeks. The dotted line at Ct value 37 indicates the cut off for YFV RNA positivity and is based on cut-offs used in the molecular diagnosis of YFV in the field. Points represent the average of two experiments where extracts from one punch were made and were fit using a linear regression.
Table 1.
Increased temperature decreases the duration of YFV RNA positivity when extracted from FTA cards.
Table 2.
Storage of FTA cards in humid environments negatively impacts the ability of the cards to stabilize YFV RNA over time.
Table 3.
FTA card desiccation prior to inoculation improves YFV RNA stabilization.