FIG. 4.

Recovery template. The recovery template (internal control) has the same sequence as the PCR product, except that the probe region has been replaced with a sequence complementary to recovery template probes. FRET detection of the target DNA is with a probe labeled with the LC-Red640 dye in channel 2 of the LightCycler instrument, while the recovery template is detected with a probe labeled with the LC-Red705 dye in channel 3. A small amount of the recovery template is added to the PCR mixture and is amplified along with the target DNA by the same primers. Thus, the two reactions compete for the primers. Normally, the recovery template is amplified from all samples, including the negative control. If neither the recovery template nor the target DNA is amplified, it is assumed that inhibition of the PCR has occurred and the test for that sample is not valid. However, if the target DNA is amplified but the recovery DNA template is not, it is assumed that the target DNA is present in a proportionally greater amount. In this situation, partial inhibition of the PCR may be present but the target DNA is successfully amplified or the recovery template may not be able to compete for primers and the recovery template signal may be weak or not present. When this occurs, the positive result is valid because the recovery template amplification is unnecessary.