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Pharmaceutical Technology BIOLOGICS AND STERILE DRUG MANUFACTURING EBOOK 2021 31 The sample is then inserted into a droplet reader, which counts the number of strongly f luorescent droplets. Finally, the software calculates the con- centration of Mycoplasma DNA in the sample using Poisson statistics. In the case of Mycoplasma detec- tion, ddPCR employs probe-based chemistry and three primers instead of two, which reduces the inci- dence of non-specific DNA amplification compared to dye-based qPCR. By quantifying Mycoplasma DNA, ddPCR technology can help AAV develop- ers accurately assess the safety of their product. Microbial detection using ddPCR technology is not a new concept. Researchers and clinicians have used ddPCR throughout the world to replace or supplement qPCR in detecting bacteria and viruses in different types of media. For instance, ddPCR has proven superior to qPCR in detect- ing pathogenic microbes in plants: scientists have used ddPCR to precisely quantify different strains of potato virus Y, an RNA virus that affects po- tato production (14), and the concentration of Spiroplasma citri, a pathogenic bacterium found in citrus fruits (15). ddPCR can also identif y Escherichia coli contamination in food, as well as detect norovirus, adenovirus, and SARS-CoV-2 in wastewater before patients present symptoms (16,17). ddPCR is also used clinically as a confir- matory step in SARS-CoV-2 testing (18). One study found that ddPCR technology de- tects bacterial species that cause serious illness in humans and animals with greater sensitivity and specificity than qPCR. To confirm the technique's robustness, researchers examined three species that project a wide range of characteristics: • Listeria monocytogenes, a gram-positive bac- terium that causes listeriosis • Francisella tularensis, a gram-negative bacte- rium that causes tularemia • Mycobacterium avium subsp. Paratuberculo- sis, a bacterial species that causes a fatal intes- tinal infection in animals and might be in- volved in causing Crohn's disease and rheumatoid arthritis in humans (19,20). All three of these species are highly potent, making it especially important that scientists can detect them at low concentrations. The re- searchers quantified all three bacterial species in suspension using both ddPCR and qPCR. They found that qPCR overestimated the quantity of each species by at least a factor of two (21). In a separate study, researchers examined the sensitivity of qPCR and ddPCR in detecting My- cobacterium tuberculosis in the blood of rhesus monkeys. While it took five weeks post-inocu- lation to detect the infection using qPCR, their ddPCR assay detected it in just two weeks (22). In a third study, researchers studied whether ddPCR could accurately quantify three different Mycoplasma species. Their chosen species repre- sent a wide range of species found in nature: • M. pneumoniae, which, as discussed previ- ously, causes bacterial pneumonia in humans • Acholeplasma laidlawii, another common cell culture contaminant • Mycoplasma hyorhinis, which causes pneu- monia in pigs. They found t hat t he limit of detection for Unlike qPCR, ddPCR technology provides an absolute count of nucleic acids, such as those found in the Mycoplasma genome.