Use of PCR analysis for sterility testing in pharmaceutical environments

To determine the sterility of pharmaceutical samples, highly conserved bact erial ribosomal DNA sequences were used in a PCR-based assay. Finished prod ucts, raw materials, growth media, and diluents were artificially contamina ted with different types of microorganisms. Samples were incubated for 24 h . After incubation, microbial DNA was extracted from enrichment broths usin g a Tris-EDTA-Tween 20 buffer containing proteinase K. Extracted DNA was ad ded to Ready-To-Go PCR beads and eubacterial primers. Contaminated samples were found to contain the conserved 1.5 kilobase (kb) DNA fragment of the b acterial genome by using the PCR assay. None of the uninoculated samples wa s found to show the presence of the 1.5 kb fragment. PCR test results were compared with standard conventional methods. There was a 100% correlation b etween standard conventional methods and the PCR assay. However, the PCR-ba sed assay was completed within 27 h while conventional methods required 4-5 days. Rapid PCR analysis using a simple sample preparation reduced the tim e for sterility testing of pharmaceutical samples allowing optimization of risk assessment and implementation of corrective actions.