Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods.
A multiplex PCR assay was devised and compared with standard conventional methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples which were artificially contaminated with <10 colony forming units of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species and possibly contaminated samples were incubated for 16 h with different enrichment media. Primers that deduce 559 bp fragment of the 16S rRNA gene was employed in amplifying E. coli species, similarly invasion protein gene with 275 bp fragment size was used as target for detecting Salmonella spp., in case of S. aureus a 461 bp amplicon from m-RNA nuclease gene, and an 709 bp fragment from oprL gene was used for amplifying P. aeruginosa. The detection limits for artificially contaminants by multiplex PCR was 1 CFU/g, where as in case of conventional method the detection limit was >2 CFU/g. Similarly, when tested with possibly contaminated samples, 35% were detected for E. coli, Salmonella spp., S. aureus and P. aeruginosa species with multiplex PCR, while only 21% were detected with standard conventional microbial methods. Multiplex PCR assay provides sensitive and reliable results and allows for the cost-effective detection of all four bacterial pathogens in single reaction tube.
Department of Microbiology, Acharya Nagarjuna University, Guntur, 522510, Andhra Pradesh, India.
Venkateswara Rao
VR
Karanam
Haranatha P
HP
Reddy
B V
BV
Subba Raju
Juvva Chandra
JC
Rao
P B
PB
Kavikishore
M
M
Vijayalakshmi
D001419:Bacteria / Q000254:growth & development / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis; D004340:Drug Contamination / Q000517:prevention & control*; D005798:Genes, Bacterial / Q000235:genetics; D007218:Industrial Microbiology / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial