Validation of Quantitative PCR Assays

can assess the safety of biotechnology products. Q-PCR precisely quantifies the amount of the nucleic acid target sequence in DNA or RNA extracted from a variety of samples including animal tissues, final products, cell banks, chromatography eluates, and bulk harvest material. During amplification with target-specific oligonucleotide primers, a fluorogenic oligonucleotide probe — with both reporter dye (FAM, VIC, TET, JOE, or HEX) and quencher dye (TAMRA) attached — anneals specifically to the amplified product between the amplimers. The detection reaction uses the 5 exonuclease activity of AmpliTaq Gold DNA polymerase (Applied Biosystems) to cleave the reporter dye from the probe, resulting in increased fluorescence when the target is present (Figure 1a). The sequence detector calculates a reporter dye fluorescence value (RN) for each PCR well during each amplification cycle. The value of RN (normalized fluorescence intensity) equals the fluorescence of the reporter dye divided by that of the passive reference dye. During PCR, RN increases as the number of amplicon copies rises until the reaction approaches a plateau. A signal baseline is established in the first few PCR cycles. Like RN, RN increases during PCR as the number of amplicon copies rises until the reaction reaches a plateau (Figure 1b). A sequence detector allows continuous measurement of the amplified product in direct proportion to the increase in fluorescence emission during PCR amplification. The threshold cycle (CT) value is calculated in real time and defined as the PCR cycle number at which an increase in reporter fluorescence (above the baseline signal) is first detected. So when the cycling point of a given amplification is first detected (where the significant increase in the fluorescence signal is associated with an exponential growth of PCR product), it represents the CT of that amplification Validation of Quantitative PCR Assays Addressing Virus Contamination Concerns