Real-Time PCR Detection ofBurkholderia cepaciain Pharmaceutical Products Contaminated with Low Levels of Bacterial Contamination
| Autor: | Theranda Jashari, Matthew Gardner, Victoria Ellman, Luis Jimenez, Margarita Kulko, Stephanie Zapata, Jenifer Vasquez, Joy Bochis, Tina Choe |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
biology Eosin methylene blue 030106 microbiology Pseudomonas Pharmaceutical Science biology.organism_classification medicine.disease_cause law.invention Microbiology 03 medical and health sciences chemistry.chemical_compound Burkholderia chemistry law SYBR Green I medicine Escherichia coli Polymerase chain reaction Bacteria Bacillus megaterium |
| Zdroj: | PDA Journal of Pharmaceutical Science and Technology. 72:73-80 |
| ISSN: | 1948-2124 1079-7440 |
| Popis: | 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 suspensions 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 methylene 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 background fluorescence was referred to as quantification cycle. All samples were found to be positive by standard microbiological testing and RT-PCR. B. cepacia was detected within 30 h 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 10 fg/uL with a correlation value of 0.98. RT-PCR detection of B. cepacia allowed faster quality control analysis, corrective actions, and process optimization. LAY ABSTRACT: A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. B. cepacia 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. B. cepacia was detected within 30 h in all contaminated samples using RT-PCR. RT-PCR detection of B. cepacia allowed faster quality control analysis, corrective actions, and process optimization. |
| Databáze: | OpenAIRE |
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