rRNA Stability in Heat-Killed and UV-Irradiated EnterotoxigenicStaphylococcus aureus and Escherichia coliO157:H7
rRNA Stability in Heat-Killed and UV-Irradiated EnterotoxigenicStaphylococcus aureus and Escherichia coliO157:H7
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DOI：
10.1128/aem.64.11.4264-4268.1998
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发表时间：
1998-11
影响因子：
4.4
通讯作者：
J. McKillip;L. Jaykus;M. Drake
中科院分区：
文献类型：
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作者：
J. McKillip;L. Jaykus;M. Drake
活细胞与非活细胞的分化在食源性病原体检测方法的发展中具有相当重要的意义。为了研究16S rRNA在核酸检测中作为细胞活力指标的适用性，我们检测了两种具有代表性的食源性致病菌——大肠杆菌O157:H7和产肠毒素金黄色葡萄球菌——在极热、中热和紫外线照射下灭活的rRNA稳定性。在37°c下孵育48小时后，在脑心灌注肉汤中生长失败，证实细胞在所有条件下均死亡。通过Northern blot分析监测rRNA的稳定性，并通过反转录(RT)- pcr对两组引物（产生325和1400 bp扩增子）进行检测评估。121℃高压灭菌15 min后，用Northern blot和RT-PCR检测两种病原菌的rRNA。相比之下，通过Northern blotting检测到两种病原体的完整rRNA，并在细胞经过80°C的热处理和254 nm的紫外线照射后48小时内可以通过RT-PCR扩增到rRNA。rRNA是在极端高温条件下监测细菌活力的合适靶分子，但在细胞中度热失活或紫外线照射后，rRNA的存在与细菌活力无关。
ABSTRACT Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens,Escherichia coli O157:H7 and enterotoxigenicStaphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37°C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1,400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121°C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80°C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.