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In drug product, the accuracy is usually validated by analysing a synthetic mixture of the drug product components which contain known amounts of a drug substance, also termed spiking or reconstituted drug product. The experimentally obtained amount of active substance is then compared to the true, added amount (recovery). It can be calculated either at each level separately as a percentage recovery or as a linear regression of the found analyte versus the added one (recovery function). Sometimes the term “recovery” is misused when reporting in active content of the drug product batches.

There are several different fluorescent detection chemistries used for real-time PCR, including the following: • SYBR® Green I, a fluorescent dye, is frequently used in real-time detection chemistry. This dye intercalates into double-stranded DNA, including PCR products and fluoresces. Therefore when used to detect amplification the level of fluorescence increases with each amplification cycle. This detection chemistry is not target sequence specific and is therefore more versatile than probe-based detection, but is susceptible to false positives due to the formation of non-specific PCR products or primer-dimers.

To note, the post-amplification laboratory can be further divided into different rooms by each detection method, depending on the number of specimens and molecular assays run by a laboratory (Figure 4). In the nested PCR laboratory second-round amplification is set-up and a thermocycler is located there for this function. Nothing from these areas should move back into the clean area, without being completely decontaminated (as described above), under any circumstances. Gloves and laboratory lab coats must be removed when leaving this area.