Molecular detection of bacterial indicators in cosmetic/pharmaceuticals and raw materials

Jiménez, Luis; Ignar, Raymond; Smalls, Stacey; Grech, Paul; Hamilton, John R.; Bosko, Yoopa; English, Donald J.

doi:10.1038/sj.jim.2900611

Cited by 38 publications

(41 citation statements)

References 7 publications

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“…Sample preparation prior to PCR analysis can be the most limiting factor during development and optimization of a given PCR assay [14]. To overcome PCR inhibition problems and to increase the sensitivity of the assay, preenrichment methods were used.…”

Section: Discussionmentioning

confidence: 99%

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

Karanam

Reddy

Raju

et al. 2008

J Ind Microbiol Biotechnol

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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.

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Section: Discussionmentioning

confidence: 99%

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

Karanam

Reddy

Raju

et al. 2008

J Ind Microbiol Biotechnol

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show abstract

“…The DNA pellet was dissolved in 50 µL of TE buffer and used for PCR assays. DNA extraction from samples inoculated by serial dilutions of S. aureus was processed as described previously (Jimenez et al. 1999).…”

Section: Methodsmentioning

confidence: 99%

“…Polymerase chain reaction (PCR) is widely accepted as a rapid and sensitive method in detection and identification of bacteria (Jimenez et al. 1999).…”

Section: Introductionmentioning

confidence: 99%

Determination of Indicator Bacteria in Pharmaceutical Samples by Multiplex PCR

Farajnia

Hassan²,

Nezhadi³

et al. 2009

Rapid Methods Automation Mic

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Rapid and sensitive detection techniques for indicator pathogens are important in pharmaceutical industry. However, common detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 5–6 days to complete. Thus, the aim of this study was to develop a multiplex polymerase chain reaction (mPCR) assay for simultaneous detection and identification of four indicator pathogenic bacteria in a single reaction. Specific primers for indicator bacteria, namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosaand Salmonella, were applied to allow simultaneous detection of them, and the sensitivity and specificity of each primer pairs were determined. In the mPCR with mixed DNA samples, specific bands for corresponding bacteria were simultaneously detected. Agarose gel electrophoresis of PCR products revealed 100% specificity of mPCR with single bands in the expected sizes. Low levels of microbial contamination less than 10 cfu per milliliter or gram of product were detected using mPCR assay. The detection of all four indicator pathogenic bacteria were completed in less than 8 h with this novel mPCR method, whereas the conventional United States Pharmacopeia methods and uniplex PCR required 5–6 days and 27 h for completion, respectively. Using mPCR assay, the microbial quality control of nonsterile pharmaceutical products can be performed in a cost‐effective and timely manner in pharmaceutical industry. PRACTICAL APPLICATIONS Detection of pathogenic indicatiors of Escherichia coli, Staphylococcus aureus, Salmonella and Pseudomonas aeruginosa is one of the mandatory tests in microbial quality of nonsterile pharmaceutical products; therefore, rapid and sensitive detection of the contaminations is of great importance for product release. According to the results of the present study, simultaneous detection of low levels of four major potential pathogenic bacteria in pharmaceutical finished products can be performed using mPCR in a cost‐effective and timely manner, and upon these properties of the mPCR assay it could have potential applications in pharmaceutical industry.

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“…Boiling method also described by Jimenez et al 18 was employed using1ml each of sample in a sterile Eppendorf tube and centrifuged at 10,000rpm for 10mins to pellet cells. The supernatant was decanted and discarded and 200µl of sterilized distilled water added to the cell pellets.…”

Section: Dna Extraction By the Boiling Methodsmentioning

confidence: 99%

“…DNA extraction using TE Buffer TE buffer method described by Jimenez et al 18 was used some modifications. 1ml of each sample was put into a sterile Eppendorf tube and then centrifuged at 10,000rpm for 10 minutes and the supernatant decanted leaving the cell pellets.…”

Section: Dna Extraction Methodsmentioning

confidence: 99%

Rapid Detection of Microbial Contamination in Ghanaian Herbal Medicines by PCR Analysis

Dei-Tutuwa¹,

Amuna²,

Rahman³

2014

Ghana Medical Journal

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Molecular detection of bacterial indicators in cosmetic/pharmaceuticals and raw materials

Cited by 38 publications

References 7 publications

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

Determination of Indicator Bacteria in Pharmaceutical Samples by Multiplex PCR

Rapid Detection of Microbial Contamination in Ghanaian Herbal Medicines by PCR Analysis

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