南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (03): 344-.doi: 10.12122/j.issn.1673-4254.2019.03.13

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大肠杆菌O157:H7 体内多聚磷酸盐的DAPI荧光定量检测

杜艳丽，韩宗利，王湘雨，万成松   

  • 出版日期:2019-03-20 发布日期:2019-03-20

A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic Escherichia coli O157:H7

  • Online:2019-03-20 Published:2019-03-20

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摘要： 目的应用荧光剂DAPI探索一种直接定量大肠杆菌O157:H7无机多聚磷酸盐（polyP）的可靠方法。方法提取并纯化大 肠杆菌O157:H7 野生株DNA，DAPI与DNA、polyP45结合，测定360 nm和415 nm激发光的发射光谱；应用共聚焦显微镜，观察 细菌DAPI-DNA和DAPI-polyP复合物荧光，验证DAPI检测polyP的可行性；细菌液氮速冻溶解、-80 ℃速冻溶解、-20 ℃速冻溶 解、60 ℃加热10 min、Triton x-100作用、室温下放置后倍比稀释涂平板，观察6种方法对细菌存活的影响；与DAPI作用后，测定 荧光值，确定细胞膜通透性的最佳前处理方法；建立标准曲线，测定EHEC野生株、ppk1 两个突变株polyP 含量。结果应用 360 nm 激发波长，DAPI-DNA最大发射波长为460 nm；应用415 nm 激发波长，DAPI-polyP 最大发射波长为550 nm；应用 405 nm激发光，收集DAPI-DNA蓝色荧光（425~475 nm），应用488 nm激发光，收集DAPI-polyP绿色荧光（500~560 nm）；最佳 细菌前处理方法为-80 ℃速冻后室温下自然溶解；标准曲线为Y=1849X+127.5（R2=0.991），测定各株菌polyP量，其中野生株显 著高于ppk1缺失突变株。结论DAPI荧光定量检测方法具有直接、可靠、易于操作、定量检测等特点，可为进一步研究polyP在 肠出血型大肠埃希菌O157:H7中的作用提供技术支持。

Abstract: Objective To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7. Methods The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃ , and freezing at -20 ℃ , all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two ppk1 mutant strains. Results At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was Y=1849X+127.5 (R2=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with ppk1 deletion. Conclusion We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.