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30 Pharmaceutical Technology BIOLOGICS AND STERILE DRUG MANUFACTURING EBOOK 2021 P h a r mTe c h . c o m of DNA. Not only does this make qPCR results relative, but it also causes measurements to vary substantially. Standard cur ves must be gener- ated manually using serial dilutions, a process that is often subject to human error. In addition, the methods used to establish the known quan- tity of the stardaed DNA are imprecise, which can ultimately cause variation in the methods and results that rely on this standard. Addition- ally, some qPCR tests that use SYBR dyes often produce false positives, leading manufacturers to overestimate the Mycoplasma concentration in their sample (13). Furthermore, qPCR measures total Mycoplasma DNA in a sample, whether it is from a living cell or simply free-floating in solution. To determine how many genome copies are derived from living cells, researchers need to measure the ratio of genome copies (GCs) and colony-forming units (CFUs). However, the GC:CFU ratio varies between cul- tures because of variable growth rates and the specific culture conditions. Therefore, researchers need to obtain an absolute count of genome copies to determine the ratio most accurately. qPCR does not have this capability as it provides relative mea- surement instead of absolute measurement. Droplet digital PCR as a Mycoplasma detection tool One technology that overcomes many of qPCR's limitations is ddPCR technology. Unlike qPCR, ddPCR technology provides an absolute count of nucleic acids, such as those found in the Myco- plasma genome. ddPCR technology employs a partitioning tech- nique that enables a researcher to measure nucleic acid concentration digitally (Figure 1). First, a scientist loads a DNA sample into a droplet gen- erator, which uses water–oil emulsification tech- nology to divide the sample into approximately 20,000 uniform 1-nL droplets, each containing one or a few nucleic acid strands. The solution enters a thermocycler, where PCR takes place separately in each droplet. These reactions use a probe that targets a particular DNA sequence, such as a piece of the Mycoplasma genome. If a droplet contains the target sequence, the probe will be cleaved as the DNA amplifies, and a re- porter dye will emit a f luorescent signal. Droplets that do not contain the target sequence will only emit weak f luorescence. Analytics Figure 1. Analog methods for nucleic acid quantification provide relative measurements of a target in a bulk sample, whereas digital methods that partition individual genome copies into droplets prior to measurement provide a direct count. FIGURE IS COURTESY OF THE AUTHOR.