RT Journal Article
SR Electronic
T1 Direct detection of Burkholderia cepacia in susceptible pharmaceutical products using semi-nested PCR
JF PDA Journal of Pharmaceutical Science and Technology
JO PDA J Pharm Sci Technol
FD Parenteral Drug Association (PDA)
SP pdajpst.2015.006049
DO 10.5731/pdajpst.2015.006049
A1 Attia, Mohamed
A1 Ali, Amal Emad ElDin
A1 Essam, Tamer M
A1 Amin, Magdy Ali
YR 2016
UL http://journal.pda.org/content/early/2016/01/14/pdajpst.2015.006049.abstract
AB Burkholderia cepacia (B. cepacia) has recently received a considerable attention as one of the major risks in susceptible pharmaceutical products. This microorganism can easily propagate and cause vast and severe contamination especially to the water supplies for pharmaceutical companies. Moreover, it proliferates within the products and can cause severe infections for humans. Therefore, fast and sensitive detection of these bacteria is of a great demand. The present study introduces improved application of PCR assay with relatively high sensitivity and specificity for the direct detection of B. cepacia from the aqueous pharmaceutical products. A semi-nested PCR (SN-PCR) approach using the primer set BCR1/BCR2 followed by BCR1/Mr yielding a 465-bp fragment of the recA gene was applied and tested using both crude lysate from isolated colonies and DNA directly extracted from artificially prepared and spiked syrup. The PCR assay showed no interference with other bacterial reference and environmental strains tested including: Staphylococcus aureus ATCC® 6538, Pseudomonas aeruginosa ATCC® 9027, Escherichia coli ATCC® 8739, Salmonella abony NCTC® 6017 and Bacillus subtilis ATCC® 6633, Micrococcus luteus, Staphylococcus warneri, Pseudomonas fluorescens, Pseudomonas putida and Ralstonia pickettii. Moreover, this semi-nested assay showed detection limit of around 10 cfu/sample and could detect B. cepacia strains isolated from municipal pre- treated potable water tank. Comparing the results for detection of B. cepacia in one hundred of randomly collected commercial syrup preparations using both conventional standard method and PCR assay revealed that, B. cepacia was detected in 2 samples using PCR assay while all samples showed negative results by conventional culturing and biochemical methods. These results highlights the advantage of using this PCR assay to detect B. cepacia in contaminated pharmaceutical products and even water for pharmaceutical purposes, without the need of culturing or pre- enrichment, where it may give false negative results and may be misidentified when biochemically tested.