Real-Time PCR Detection of in Pharmaceutical Products Contaminated with Low Levels of Bacterial Contamination.

A real-time polymerase chain reaction (RT-PCR) assay was developed to detect in pharmaceutical products contaminated with low levels of bacteria. Different pharmaceutical suspensions were artificially contaminated with , , , and 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 Selective media isolation of bacterial contamination showed only detected on pseudomonas isolation while eosin methylene blue and MacConkey detected only 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 background fluorescence was referred to as quantification cycle. All samples were found to be positive by standard microbiological testing and RT-PCR. was detected within 30 h in all contaminated samples using RT-PCR. Based upon standard curve analysis of DNA, the minimum DNA concentration that could be detected was 10 fg/uL with a correlation value of 0.98. RT-PCR detection of allowed faster quality control analysis, corrective actions, and process optimization. A real-time polymerase chain reaction (RT-PCR) assay was developed to detect in pharmaceutical products contaminated with low levels of bacteria. is the number one reason for microbial contamination recalls of non-sterile drug products in the USA. 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. All samples were found to be positive by standard microbiological testing and RT-PCR. was detected within 30 h in all contaminated samples using RT-PCR. RT-PCR detection of allowed faster quality control analysis, corrective actions, and process optimization.