Abstract
A Real-Time (RT) PCR assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacterial contamination. Different pharmaceutical suspensions were artificially contaminated with B. cepacia, Escherichia coli, Staphylococcus aureus, and Bacillus megaterium. After a 24-hour incubation in TSB 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 PIA while EMB and MAC 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 background fluorescence was referred as crossing point (Cp). All samples were found to be positive by standard microbiological testing and RT-PCR. B. cepacia was detected within 30 hours in all contaminated samples using RT-PCR. Based upon standard curve analysis of B. cepacia DNA, the minimum DNA concentration that could be detected was 9.96 x 10-6 ng/ul with a correlation value of 0.98. RT-PCR detection of B. cepacia allowed faster quality control analysis, corrective actions, and process optimization.
- Received June 14, 2017.
- Accepted October 3, 2017.
- Copyright © 2017, Parenteral Drug Association
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