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首页> 外文期刊>Biotechnology Progress >A Recombinant Bacteriophage-Based Assay for the Discriminative Detection of Culturable and Viable but Nonculturable Escherichia coli O157:H7
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A Recombinant Bacteriophage-Based Assay for the Discriminative Detection of Culturable and Viable but Nonculturable Escherichia coli O157:H7

机译：基于重组噬菌体的可鉴别和可培养但不可培养的大肠杆菌O157：H7的鉴别检测

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摘要

A previously green fluorescent protein (GFP)-labeled PP01 virulent bacteriophage,specific to Escherichia coli O157:H7,was used to construct lysozyme-inactivated GFP-labeled PP01 phage (PP01e~-/GFP).The new recombinant phage lacked lytic activity because of the inactivation of gene e,which produces the lysozyme responsible for cell lysis.Gene e was inactivated by inserting an amber stop codon.Prolonged incubation of E.coli O157:H7 cells with PP01e~-/GFP did not lead to cell lysis,while the propagation of PP01e~-/GFP in host cells increased the intensity of green fluorescence.Retention of cell morphology and increase in fluorescence enabled the direct visualization and enumeration of E.coli O157:H7 cells within an hour.The PP01e~-7GFP system,when combined with nutrient uptake analysis,further allowed the discriminative detection of culturable,viable but nonculturable (VBNC),and dead cells in the stress-induced aquatic environment.Stress-induced cells,which retained culturability,allowed phage propagation and produced bright green florescence.Nonculturable cells (VBNC and dead)allowed only phage adsorption but no proliferation and remained low fluorescent.The low-fluorescent nonculturable cells were further differentiated into VBNC and dead cells on the basis of nutrient uptake analysis.The low-fluorescent cells,which grew in size by nutrient incorporation during prolonged incubation in nutrient medium,were defined as metabolically active and in the VBNC state.The elongated VBNC cells were then easily recognizable from dead cells.The proposed assay enabled the detection and quantification of VBNC cells.Additionally,it revealed the proportion of culturable to VBNC cells within the population,as opposed to conventional techniques,which demonstrate VBNC cells as a differential value of the total viable count and the culturable cell count.
机译：以前用绿色荧光蛋白（GFP）标记的PP01毒性噬菌体特异于大肠杆菌O157：H7，用于构建溶菌酶灭活的GFP标记的PP01噬菌体（PP01e〜-/ GFP）。新的重组噬菌体缺乏裂解活性，因为基因e失活，产生负责细胞裂解的溶菌酶。基因e通过插入琥珀色终止密码子而失活。大肠杆菌O157：H7细胞与PP01e〜-/ GFP长时间孵育不会导致细胞裂解，而PP01e〜-/ GFP在宿主细胞中的繁殖增加了绿色荧光的强度。细胞形态的保留和荧光的增加使得一小时内可直接观察和计数O157：H7大肠杆菌。PP01e〜-7GFP系统中，结合营养吸收分析，可以判别检测在压力诱导的水生环境中可培养的，存活的但不可培养的（VBNC）和死细胞。应力诱导的细胞可保留可培养性噬菌体繁殖并产生明亮的绿色荧光。非培养细胞（VBNC和死细胞）仅允许噬菌体吸附但没有增殖，并保持低荧光。根据养分吸收分析，低荧光的不可培养细胞进一步分化为VBNC和死细胞。低荧光细胞是指在营养培养基中长时间孵育后由于营养物质的掺入而增长的大小，被定义为具有代谢活性且处于VBNC状态。细长的VBNC细胞易于从死细胞中识别出来。此外，它揭示了群体中可培养细胞对VBNC细胞的比例，这与传统技术相反，传统技术表明VBNC细胞是总生存数与可培养细胞数的差值。

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