| 摘要: | 化粧品中常見的污染菌有大腸桿菌（Escherichia coli）、金黃色葡萄球菌（Staphylococcus aureus）、綠膿桿菌（Pseudomonas aeruginosa）、炫饃黖腄]Bacillus subtilis）和白色念珠菌（Candida albican）。本研究應用聚合酶鏈反應（Polymerase Chain Reaction, PCR）技術快速檢驗化粧品中，是否有受E. coli、S. aureus和P. aeruginosa之污染。研究中選用此三株菌共有的一種基因DNA Polymerase III（delta subunit）設計出三組引子，此三組引子在個別或混合E. coli、S. aureus、P. aeruginosa、B. subtilis和C. albican 的基因體DNA進行PCR反應後，結果顯示EC2（+）／EC1（-）引子於黏合溫度60℃下，會專一性地對E. coli產生643 bp的產物；SA3（+）／SA3（-）引子於黏合溫度60℃下，會專一性地對S. aureus產生510 bp的產物；PA2（+）／PA2（-）引子於黏合溫度67℃下，會專一性地對P. aeruginosa產生365 bp的產物。以菌體當模板直接進行PCR反應後的結果顯示，此三組引子仍可專一性地得到上述的三種產物大小，故此檢驗技術中的酵素反應不受菌體干擾，可省略DNA萃取之步驟。另外，此三組引子在同屬不同種的Escherichia vulneris、Staphylococcus epidermidis和Pseudomonas fluorescens之間進行PCR反應後的結果顯示，此三組引子並不會產生PCR之DNA產物。在PCR檢驗技術的偵測極限方面，三組引子皆為10的5次方CFU/mL。將此法模擬至受E. coli、S. aureus、P. aeruginosa、B. subtilis和C. albican污染的保濕化粧水檢驗上所得到的結果，分別偵測到643、510和365 bp三個產物大小，這表示此法可清楚地辨識出受微生物污染的保溼化粧水中是否有受此三株菌之污染。最後將PCR的產物以自動定序法確認DNA序列，再與GenBank資料庫之基因序列作比對後，結果顯示E. coli (Migula) Castellani and Chalmers（BCRC 11509）的PCR產物與E. coli CFT073完整的基因體序列有99.7％的相同度；S. aureus subsp. aureus Rosenbach（BCRC 10451）與S. aureus subsp. aureus USA有100.％的相同度；P. aeruginosa (Schroeter) Migula（BCRC 11864）與P. aeruginosa UCBPP-PA14有99.2％的相同度。
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. |
