文章摘要
汪河,王莉,申慧彦,李卫华.水质细菌总数FISH检测技术的优化[J].安徽建筑大学学报,2022,30(1):46-54
水质细菌总数FISH检测技术的优化
Optimization of Fluorescence in Situ Hybridization (FISH) for the Total Bacterial Count in Water
  
DOI:
中文关键词: 水质细菌总数  FISH技术  优化  稳定性
英文关键词: total bacterial count in water  FISH technology  optimization  stability
基金项目:国家自然科学基金项目(51978003);安徽省重点研发项目(1704a0902006);安徽省科技重大专项(17030801028)
作者单位
汪河 安徽建筑大学  环境与能源工程学院安徽  合肥  230601 环境污染控制与废弃物资源化利用安徽省重点实验室安徽  合肥  230601 
王莉 安徽建筑大学  环境与能源工程学院安徽  合肥  230601 环境污染控制与废弃物资源化利用安徽省重点实验室安徽  合肥  230601 
申慧彦 安徽建筑大学  环境与能源工程学院安徽  合肥  230601 环境污染控制与废弃物资源化利用安徽省重点实验室安徽  合肥  230601 
李卫华 安徽建筑大学  环境与能源工程学院安徽  合肥  230601 环境污染控制与废弃物资源化利用安徽省重点实验室安徽  合肥  230601 
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中文摘要:
      在水质卫生检测方面,FISH 具有检测快速、可检测指标多样、检出微生物数量准确等优势。但因其 结果稳定性较差,尚不能替代培养计数法成为标准方法。本研究从影响结果稳定性的细胞制片、蛋白酶 K 消 化时间、探针浓度和杂交时间四个方面优化 FISH 技术。结果表明,46 ℃、2 h 烘烤制片可明显减少因洗涤造 成的细胞流失;终浓度 10 μg/mL 的蛋白酶 K 经 37 ℃、2 min 消化可使样品得到较高的荧光信号且背景干扰少; 探针终浓度 5 ng/μL,46 ℃、5 h 的条件下可促进寡核苷酸探针 EUB338 mix 与细菌细胞充分杂交。优化后的 FISH 定量结果较稳定,三次实验的荧光细胞数量无明显差异(P>0.05)。本研究为水质细菌总数检测的 FISH 法走向标准化提供了一定的技术基础。
英文摘要:
      Fluorescence in Situ Hybridization (FISH) is a water testing technique with the advantages of rapid detection,various detectable indexes,and accurate detection of microbial quantities. However,its results are not stable enough to replace the counting method as a standard method. In this study,the FISH technique was optimized in four aspects,namely,cell sample preparation, proteinase K digestion time,probe concentration and hybridization time,which affect the stability of the results. The results showed that the washing-caused cell loss could be significantly reduced by baking the glass slides at 46 ℃ for 2 hours. The digestion of proteinase K at a final concentration of 10 μg/mL at 37 ℃ for 2 minutes could result in a high fluorescence signal with less background interference. The probe final concentration of 5 ng/μL at 46 ℃ for 5 hours could facilitate the hybridization of the oligonucleotide probe EUB338-mix and bacterial cells. The optimized FISH technique showed good stability,and there was no significant difference in the number of fluorescent cells among the three experiments (P>0. 05). This study provides a technical basis for the standardization of FISH method for the detection of total bacterial count in water.
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