Page 69 - 南京医科大学学报自然科学版

第41卷第6期 南京医科大学学报（自然科学版）
2021年6月 Journal of Nanjing Medical University（Natural Sciences） ·847 ·

·基础医学·

基于叠氮溴化丙锭结合高通量测序技术分析口腔综合治疗台

水路系统中活菌多样性的研究

周如玉 ，平逸帆 ，张 元 ，王 娟 1，2*
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1 南京医科大学口腔疾病研究江苏重点实验室，江苏 南京 210029；南京医科大学附属口腔医院牙体牙髓病科，正畸科，江
3
2
苏 南京 210029

［摘 要］ 目的：利用叠氮溴化丙锭（propidium monoazid，PMA）预处理方法结合高通量测序技术检测口腔综合治疗台水路系
统（dental unit waterline，DUWL）中活菌微生物群落，以综合评估不同科室 DUWL 中的污染情况及探究活菌群落构成的多样
性。方法：选择南京医科大学附属口腔医院33台口腔综合治疗台，牙体牙髓病科23台（E组），牙周病科10台（P组），在医院开
诊前收集三用枪水样，PMA预处理水样后提取活菌总DNA，经细菌通用引物PCR扩增后构建宏基因组文库，用于高通量测序
和生物信息学分析。结果：样本使用 1 μL PMA（最终浓度 20 μmol/L）预处理 10 min 后暴露于卤素光照射 5 min，最适合检测
DUWL样品中活菌的量；高通量测序显示DUWL中活菌微生物群落呈多样性，并具有潜在的细菌致病序列。科室间具有统计
学差异的菌门为蓝藻菌门（Cyanobacteria）、放线菌门（Actinobacteria）、衣原体门（Chlamydiae）、Aerophobetes、绿弯菌门（Chloro⁃
flexi）、Bacteria_d_norank_k__unclassified、TM6（P < 0.05）；属水平上，军团菌属（Legionella）、Methyloversatilis、Obscuribacterales__
norank差异有统计学意义（P < 0.05）。结论：DUWL中致病微生物的存在对牙科医务工作者和患者都具有潜在感染风险，应重
视DUWL细菌群落的多样性，有必要对DUWL进行定期水质控制。
［关键词］ 叠氮溴化丙锭；高通量测序；口腔综合治疗台水路系统；微生物多样性
［中图分类号］ R780.2 ［文献标志码］ A ［文章编号］ 1007⁃4368（2021）06⁃847⁃09
doi：10.7655/NYDXBNS20210609

Analysis on viable bacterial diversity in dental unit waterlines based on propidium
monoazid in conjunction with high⁃throughput sequencing

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ZHOU Ruyu ，PING Yifan ，ZHANG Yuan ，WANG Juan 1，2 *
1 Jiangsu Key Laboratory of Oral Diseases of Nanjing Medical University，Nanjing 210029；Department of
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Endodontics，Department of Orthodontics，the Affiliated Stomatological Hospital of Nanjing Medical University，
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Nanjing 210029，China
［Abstract］ Objective： In order to evaluate the microbial contamination of different departments and explore the diversity of living
bacterial community，the method of propidium monoazid（PMA）pretreatment combined with high⁃throughput sequencing technology
was used to detect the living microbial community in the dental unit waterline（DUWL）. Methods：A total of 33 dental chair units
（DCU）were selected from the Affiliated Stomatological Hospital of Nanjing Medical University，with 23 from endodontics department
（group E）and 10 from periodontics department（group P）. Before patients arrived，water samples were collected from each DCU using
a three⁃in⁃one air/water syringe. After PMA pretreatment，the total DNA of living bacteria was extracted. After PCR amplification with
bacterial universal primers，the macrogenomic library was constructed for high⁃throughput sequencing and bioinformatics analysis.
Results：Samples were pretreated with 1 μL of PMA（20 μmol/L）for 10 min and subsequently exposed to light irradiation for 5 min，
which were optimal for detecting viable bacterial in samples of DUWL. The output water of DUWL was associatied with a high bacterial
diversity and identified potential bacterial pathogenic sequence. The statistically different phyla between the two groups were
Cyanobacteria，Actinobacteria，Chlamydiae，Aerophobetes，Chloroflexi，Bacteria_d_norank_k__unclassified，TM6（P < 0.05）. The

［基金项目］ 南京医科大学2017校基金（2017NJMU112）；南京医科大学校基金（NMUB2019073）
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通信作者（Corresponding author），E⁃mail： candy1249@126.com