| Summary: | 碩士 === 嘉南藥理科技大學 === 化妝品科技研究所 === 96 === The common contamination of microorganism in cosmetic products include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Candida albican. In this study, we develop a rapid method to detect E. coli, S. aureus and P. aeruginosa in cosmetic by PCR technique. We design three pairs of specific primer for DNA Polymerase III (delta subunit) gene of E. coli, S. aureus and P. aeruginosa. Single or mixed genomic DNA of E. coli, S. aureus, P. aeruginosa, B. subtilis and C. albican were examined in this PCR technique. These results exhibit EC2(+)/1(-) primers specifically produce a DNA fragment of 643bp at annealing temperature 60℃ for E. coli, and SA3(+)/3(-) primers specifically make a fragment of 510bp at annealing temperature 60℃ for S. aureus than PA2(+)/2(-) primers specifically result a PCR product of 365bp at annealing temperature 67℃ for P. aeruginosa. We also found that microorganisms did not influence the result of PCR by adding colonies into the detective reaction directly. Furthermore, the three pairs of specific primer didn’t make any PCR products in Escherichia vulneris, Staphylococcus epidermidis and Pseudomonas fluorescens. On the limitation of PCR for the detection of microorganisms, our results all show in 105 CFU/mL. We also use this technique to detect the moist lotion that contains E. coli, S. aureus, P. aeruginosa, B. subtilis and C. albican. The result reveals that the technique can rapidly and accurately detect E. coli, S. aureus and P. aeruginosa in the contaminative moist lotion. Ultimately, we confirmed these sequences of PCR products by autosequencing and compared them with other genes in GenBank. Our results respectively display that the PCR product of E. coli (Migula) Castellani and Chalmers, BCRC 11509, has 99.7 % identity with E. coli CFT073, and the PCR product of S. aureus subsp. aureus Rosenbach, BCRC 10451, has 100.% identity with S. aureus subsp. aureus USA then the PCR product of P. aeruginosa (Schroeter) Migula, BCRC 11864, has 99.2% identity with P. aeruginosa UCBPP-PA14.
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