大肠杆菌在lacZ基因表达和调控方面与气单胞菌、假单胞菌的异同及膜荧光法检测时“假阳性”的克服

Difference in Expression in lacZ Gene and Regulation of Escherichia Coli from Aeromonas Hydrophila and Pseudomonas Aeroginosa and Overcoming the False-positive Reaction while Detecting by Filter Membrane Fluorometry

目的：探讨气单胞菌、假单胞菌在ｌａｃＺ基因表达调控方面与大肠菌群的差异，提高“大肠菌群产荧光底物试验”的准确性。方法：以ＭＵＧＡＬ作为β－半乳糖苷酶（β－ＧＡＤ）的产荧光反应底物。配制底物、诱导物、葡糖不同组合的３种培养液，对各试验菌在培养液中荧光强度Ｆ值变化进行测定，比较其ｌａｃＺ基因表达的“诱导效应”与“葡糖效应”的不同表现特点。用微孔膜捕集孤立的试验菌以含上述培养液的衬垫支持，观测比较“单个”试验菌细胞在滤膜表面形成荧光斑点的情况。结果：①大肠杆菌在乳糖诱导下能高效产生β－ＧＡＤ，Ｆ／Ｆ０＝２２．７６，而葡糖的存在可完全抑制ｌａｃＺ基因表达，Ｆ／Ｆ０＝０．９６；滤膜上单个Ｅ．ｃｏｌｉ菌可在１２～１５ｈ内形成强烈并带有扩散性“晕轮”的蓝色荧光斑点。②嗜水气单胞菌“诱导效应”与“葡糖效应”均不明显，仅产生少量β－ＧＡＤ。在滤膜上需经３０ｈ才能产生微弱的不带“晕轮”的荧光斑点。③铜绿假单胞菌能产生与β－ＧＡＤ无关的荧光色素，其光谱特性与ＭＵＧＡＬ水解的４－ＭＵ完全不同，在滤膜上表现为黄绿色无晕轮的荧光斑。结论：采用滤膜荧光法检测大肠菌群，可利用产生荧光斑点的时间差、色泽及有无晕轮的特性克服气单胞菌、假单胞菌?

Objective: The purpose of the study was to improve the accuracy of fluorescent substrate coliform test by a comparison of Escherichia coli (E.Cdi), Aeromonas hydrophila and Pseudomonas aeroginosa in expression in LacZ gene and regulation. Method:MUGAL was used as a fluorescent substrate of β -galactosidase( β -GAD) and three different mediums were mixed with substrate.The variation of fluorescence intensity of the bacteria studied was detected and the characteristic of “induction effect' and “glucose effect' of the expression in lacZ gene were compared. We collected the strains by microporous membrane on underlay in the nutrient solution and observed the fluorescent spots of single colony on the surface of filter membrane.Results: ① E.coli can produces β -GAD effectively induced by lactose, F/F 0 =22.76, and the single strain formed intense and diffused blue fluorescent spot with halo within 12～15h. But the expression in lacZ gene can be completely repressed by glucose, F/F 0 =0.96. ②Both “induction effect' and “glucose effect' of Aeromonas hydrophila were not obvious except for hydrolyzing a trifle of β -GAD. After 30h, weak fluorescent spot appeared on filter membrane.③ Pseudomonas aeroginosa can produce fluochrome spot unconcerned with β -GAD whose spectral characteristic was different from that of 4-MU hydrolyzed by MUGAL and performed olivine fluorescent spot without halo. Conclusion : The time interval of producing fluorescent spot,the color and characteristic of having “halo' or not can overcome the interference from Aeromonas hydophila and Pseudomonas aeroginosa when detecting coliforms by filter membrane fluorometry method.