Simultaneous PCR detection of bacteria and mold DNA sequences in pharmaceutical samples by using a gradient thermocycler

摘要

A gradient thermocycler, the Stratagene RoboCycler 96-Gradient, was evaluated for the simultaneous PCR amplification of microbial genes which indicated the presence of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger in pharmaceutical samples. Suspensions of pharmaceutical products were inoculated with pure cultures of bacteria and mold. After a 24 h incubation, bacterial DNA was extracted from each enrichment broth using a mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling the samples in Tris-EDTA-SDS buffer for 1 h. A 10 muL aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50 muL aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. The individual samples were loaded into the RoboCycler 96-Gradient thermocycler. Simultaneous detection of all microbial genes was performed by using a gradient profile that allowed the use of DNA primers with different annealing temperatures. Standard methods for quality control evaluation of pharmaceutical products required 6-8 days while simultaneous PCR detection of bacteria and mold DNA sequences was completed within 27 h.