Real-Time PCR Detection of Burkholderia cepacia in Pharmaceutical Prod-ucts Contaminated with Low Levels of Bacterial Contamination
Jimenez L, Jashari T, Vasquez J, et al. (2018). PDA J Pharm Sci and Tech, vol. 72, pp. 73-80.
A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. Different pharmaceutical suspen-sions were artificially contaminated with B. cepacia, Escherichia coli, Staphylococcus aureus, and Bacillus megaterium. After a 24 h incubation in trypticase soy broth with Tween 20, samples were streaked on mannitol salt, phenyl ethyl alcohol, eosin methylene blue, MacConkey, and pseudomonas isolation agar. Microbial DNA was extracted from each sample by using a Tris-EDTA, proteinase K, Tween 20 buffer. Regular PCR targeting the 1.5 kilobases 16S rRNA eubacterial gene and cloning showed the predominant DNA in the extracted mix belonged to E. coli. Selective media isolation of bacterial contamination showed B. cepacia only detected on pseudomonas isolation while eosin meth-ylene blue and MacConkey detected only E. coli. RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. The cycle at which fluorescence from amplification exceeds the back-ground fluorescence was referred to as quantification cycle.