16S rRNA coding gene PCR for the early detection of bacterial contamination of cell culture and reagents used for FMD vaccine production

M. Tech. Veterinary Technology.Ensuring that biological products (specifically vaccines) are free of bacterial contamination presents a significant challenge. The European Pharmacopoeia recommends the routine use of plates, broths and a filtration system for detection of bacterial contaminants that might be present in raw materials. However, these methods could not timeously detect slow growing bacterial contaminants and a polymerase chain reaction was developed for the early detection of bacterial contamination. To eliminate false positive results the researcher compared ethidium monoazide bromide and propidium monoazide to distinguish between viable and non-viable bacterial cells, using universal primers which amplified a ±1.5 kilobase pair fragment of 16S ribosomal ribonucleic acid. The results indicated propidium monoazide more sensitive to detect low levels of viable bacteria. A comparative test between European Pharmacopoeia recommended and polymerase chain reaction methods found that the latter were the more sensitive method for contamination detection. The sequencing results of positive samples indicated the following contaminants: Stenotrophomonas spp. and Pseudomonas spp. as pre-dominant followed by Bacillus spp., Enterobacter spp., Klebsiella spp. and Propionibacterium spp. Propidium monoazide combined with polymerase chain reaction showed promise as a rapid, reproducible method for accessing cell integrity and is valuable to those who wish to employ it as an early detection method of possible contamination within a production system