| 汪河,王莉.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(): |
| 水质细菌总数FISH检测技术的优化 |
| Optimization of Quantitative Processing of Fluorescence in Situ Hybridization (FISH) for Total Bacterial Count in Water Quality |
| 投稿时间：2021-03-10 修订日期：2021-03-31 |
| 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: optimization of quantitative processing FISH technology optimization stability |
| 基金项目:国家自然科学基金（51978003）；安徽省重点研发项目（1704a0902006）；安徽省科技重大专项（17030801028） |
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| 中文摘要: |
| 在水质卫生检测方面，FISH具有快速、能同时检测多种指标、检测出的微生物数量更接近实际等优势。但，因其结果稳定性较差，目前尚不能替代培养计数法成为标准方法。本研究在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对FISH技术进行优化。结果表明，46℃ 2h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 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，可使寡核苷酸探针EUB338 mix与细菌细胞充分地杂交。优化后的FISH定量结果稳定性较好，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: |
| In terms of water quality and sanitation detection, Fluorescence in Situ Hybridization (FISH) has the advantages of fast, simultaneous detection of multiple indicators, and the number of microorganisms detected is closer to the actual situation. However, it has not replaced the culture counting method as the standard method because of its poor stability. In this study, we optimized FISH technology in four aspects, cell immobilization on glass slide, protease K digestion time, probe action concentration and hybridization time. The results showed that the cell loss caused by washing could be significantly reduce by baking slides with cells at 46 ℃ for 2h. The higher fluorescence signal and less background interference were acquired with 10 μg/ml of proteinase K digesting samples at 37 ℃ for 2 min. When the final concentration of probe was 5 ng/μL and the temperature was 46 ℃, it was observed at 5 h that bacterial cells could be fully hybridized with the oligonucleotide probe EUB338 mix. The optimized FISH technology 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 bacterial count detection in water quality. |
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