FMDV

Reverse transciptase loop-mediated isothermal amplification was used to detect three of the six serotypes of FMDV (O, A, and Asia-1). Three pairs of primers are required for each LAMP reaction. For all three serotypes the primers targeted the 3D polymerase gene (primer sequences included in methods). The reaction uses reverse transcriptase and DNA polymerase and runs at 60℃ for up to 1 hour. Presence of viral RNA was visualized with Hydroxy napthol blue which changes from purple to blue for a positive result. RT-LAMP was shown to be more sensitive than conventional PCR and comparable to real time PCR.

RT-LAMP and RT-LAMP with added swarm primers (sRT-LAMP) were compared to traditional PCR and previous attempts at RT-LAMP for identification of the O serotype for FMDV. They targeted the p73 gene with both RT-LAMP and sRT-LAMP. Using p73 as the target yielded more accurate positives than did targeting p72 as previous iterations of RT-LAMP had done. RT-LAMP requires 3 pairs of primers and sRT-LAMP adds an additional pair meant to improve specificity. Both reactions are optimized at 6℃ for 40 min but can be run anywhere between 59-68℃. Color change by hydroxy napthol blue was used to indicate presence of viral RNA. RT-LAMP and sRT-LAMP both had excellent specificity for the O serotype of FMDV. Neither gave positive readings for other FMDV serotypes so these assays with these sets of primers are useful for identifying only the O serotype.

RT-PCR was optimized for FMDV of all serotypes. Primers developed by Callahan, et al. (2002) were highly specific to FMDV and targeted all four of the sero-types tested including SAT-1 and SAT-2. Field samples were taken from cattle in East Africa where serotpes O, A, SAT-1, and SAT-2 were all found.

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