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Research ArticleTechnology/Application

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

Luis Jimenez, Theranda Jashari, Jenifer Vasquez, Stephanie Zapata, Joy Bochis, Margarita Kulko, Victoria Ellman, Matthew Gardner and Tina Choe
PDA Journal of Pharmaceutical Science and Technology January 2018, 72 (1) 73-80; DOI: https://doi.org/10.5731/pdajpst.2017.007971
Luis Jimenez
Biology and Horticulture Department, Bergen Community College, Paramus, NJ
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  • For correspondence: papojbmicro@gmail.com
Theranda Jashari
Biology and Horticulture Department, Bergen Community College, Paramus, NJ
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Jenifer Vasquez
Biology and Horticulture Department, Bergen Community College, Paramus, NJ
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Stephanie Zapata
Biology and Horticulture Department, Bergen Community College, Paramus, NJ
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Joy Bochis
Biology and Horticulture Department, Bergen Community College, Paramus, NJ
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Margarita Kulko
Biology and Horticulture Department, Bergen Community College, Paramus, NJ
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Victoria Ellman
Biology and Horticulture Department, Bergen Community College, Paramus, NJ
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Matthew Gardner
Biology and Horticulture Department, Bergen Community College, Paramus, NJ
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Tina Choe
Biology and Horticulture Department, Bergen Community College, Paramus, NJ
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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. 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.

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PDA Journal of Pharmaceutical Science and Technology: 72 (1)
PDA Journal of Pharmaceutical Science and Technology
Vol. 72, Issue 1
January/February 2018
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Real-Time PCR Detection of Burkholderia cepacia in Pharmaceutical Products Contaminated with Low Levels of Bacterial Contamination
Luis Jimenez, Theranda Jashari, Jenifer Vasquez, Stephanie Zapata, Joy Bochis, Margarita Kulko, Victoria Ellman, Matthew Gardner, Tina Choe
PDA Journal of Pharmaceutical Science and Technology Jan 2018, 72 (1) 73-80; DOI: 10.5731/pdajpst.2017.007971

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Real-Time PCR Detection of Burkholderia cepacia in Pharmaceutical Products Contaminated with Low Levels of Bacterial Contamination
Luis Jimenez, Theranda Jashari, Jenifer Vasquez, Stephanie Zapata, Joy Bochis, Margarita Kulko, Victoria Ellman, Matthew Gardner, Tina Choe
PDA Journal of Pharmaceutical Science and Technology Jan 2018, 72 (1) 73-80; DOI: 10.5731/pdajpst.2017.007971
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