Fig 1.

The engineered control material allows differential amplification of RNA and DNA from transgenic organisms. The bacterium of interest is transformed with a plasmid vector containing a target gene construct, wherein the target gene is disrupted by an autocatalytic, self-splicing intron that has activity in the chosen organism. Primers are designed that specifically amplify the spliced RNA transcript, rather than the template DNA, either by binding across the exon–exon junction (top primer) or because the amplicon length is prohibitively large, unless the intron has first been successfully excised (bottom primer). This schema allows selective amplification of RNA from the transgenic organism, which can be used to monitor successful assay performance through all phases of testing (bacterial lysis, nucleic acid recovery, reverse transcription, template amplification, and signal detection).