Detection of Escherichia coli ATCCRTM 8739(TM) and Aspergillus brasiliensis ATCCRTM16404(TM) in Raw Materials and Pharmaceutical Products Using the Real-Time PCR in Comparison with Standard Conventional Microbiological Methods

Pharmaceutical products are susceptible to microbial contamination. This type of contamination could represent a risk to consumers health. Furthermore, microbial contamination can cause degradation, changes in the aesthetic of the product, and loss of drug effectiveness by reducing or inactivate the therapeutic activity of the product. The techniques used in the pharmaceutical industry are conventional techniques where the practice of methods of transference of cultures, phenotypic observation of the colony, and biochemical tests for its final identification prevails. These conventional techniques are time-consuming, not specific, and lack accuracy and precision to demonstrate the present of specified organisms in a sample. This analytical methodology results in delays for the final approval of products. A sensitive Real-time Polymerase Chain Reaction method with TaqManRTM MGM probe was developed in this research for precision, specificity and rapid detection of objectionable microorganisms in raw material and finished product. The Real-time PCR method had amplification of Escherichia coli ATCCRTM 8739(TM) DNA in all dilutions from 10¯¹ to 10¯¹5mL by the sensitivity test and detected the bacteria in the raw materials and OTC samples analyzed at 10¯7 and 10¯¹5mL for Test for Specified Microorganisms (TSM). Also, in Aspergillus brasiliensis ATCCRTM16404(TM) was obtained amplification in all samples from sample 10¯¹ to 10¯¹5mL for sensitivity test and 10¯³ and 10¯¹5mL for detection of the presence of the fungi in TSM. There is a statistically significant differences in the detection of Escherichia coli ATCCRTM 8739(TM) and ATCCRTM 16404(TM) Aspergillus brasiliensis in conventional and the rt-PCR methods. The conventional method did not have the ability to detect small traces of the 10¯¹5 sample dilutions while the Real-time PCR method was able to detect at this dilution. This research demonstrates how quickly, precisely, and accurately detect the presence of these objectionable organisms in terms of the minimum traces as 4 mul of pharmaceutical sample, something not possible under the conventional USP pour plate method. The Real-time PCR methodology contributes to the rapid detection of objectionable organisms in a pharmaceutical sample preventing the risk of exposure of humans and animals to contaminated drugs that do not meet the FDA and USP quality standards.