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强氯精对对虾养殖用水中抗生素抗性基因的影响

夏涛涛 苏浩昌 胡晓娟 徐煜 文国樑 曹煜成 余招龙

夏涛涛, 苏浩昌, 胡晓娟, 徐煜, 文国樑, 曹煜成, 余招龙. 强氯精对对虾养殖用水中抗生素抗性基因的影响[J]. 南方水产科学, 2022, 18(6): 85-92. doi: 10.12131/20210361
引用本文: 夏涛涛, 苏浩昌, 胡晓娟, 徐煜, 文国樑, 曹煜成, 余招龙. 强氯精对对虾养殖用水中抗生素抗性基因的影响[J]. 南方水产科学, 2022, 18(6): 85-92. doi: 10.12131/20210361
XIA Taotao, SU Haochang, HU Xiaojuan, XU Yu, WEN Guoliang, CAO Yucheng, YU Zhaolong. Effect of trichloroisocyanouracic acid on antibiotic resistance genes in aquaculture water of shrimp[J]. South China Fisheries Science, 2022, 18(6): 85-92. doi: 10.12131/20210361
Citation: XIA Taotao, SU Haochang, HU Xiaojuan, XU Yu, WEN Guoliang, CAO Yucheng, YU Zhaolong. Effect of trichloroisocyanouracic acid on antibiotic resistance genes in aquaculture water of shrimp[J]. South China Fisheries Science, 2022, 18(6): 85-92. doi: 10.12131/20210361

强氯精对对虾养殖用水中抗生素抗性基因的影响

doi: 10.12131/20210361
基金项目: 广东省重点领域研发计划 (2021B0202040001);国家现代农业产业技术体系 (CARS-48);广东省自然科学基金项目 (2019A1515011618);广东省现代农业产业技术体系创新团队 (2019KJ149);中国水产科学研究院基本科研业务费专项资金 (2020TD54, 2020XK02);中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金资助 (2021SD08)
详细信息
    作者简介:

    夏涛涛 (1995—),男,硕士研究生,研究方向为养殖环境安全。E-mail: 1903096289@qq.com

    通讯作者:

    曹煜成 (1979—),男,研究员,博士,研究方向为水产健康养殖和养殖生态环境调控。E-mail: cyc_715@163.com

  • 中图分类号: S 949

Effect of trichloroisocyanouracic acid on antibiotic resistance genes in aquaculture water of shrimp

  • 摘要: 为探讨渔用氧化剂——强氯精 (三氯异氰脲酸,C3Cl3N3O3) 对养殖水体环境中抗生素抗性基因 (Antibiotic resistant genes, ARGs) 的去除并控制其传播的可行性,采用实时荧光定量PCR技术,分别对近海水源水、蓄水沉淀池水体、氧化消毒后养殖备用水中的ARGs浓度进行为期29 d的监测分析,其中氧化消毒时的强氯精使用质量浓度为40 mg·L−1。测定的ARGs包括sul1sul2tetXtetMfloRcmlAqnrA 7种养殖环境中常见的ARGs。结果显示,sul1sul2floRtetX为上述3种水样中的优势ARGs。近海水源水中ARGs的种类数量及总浓度均最高;蓄水沉淀池水体中ARGs的总浓度低于水源水,sul2floR的浓度分别较水源水降低了0.86和0.34 lg;经氧化消毒后水体中ARGs的总浓度也有所下降,sul2floR的浓度与水源水相比分别降低了1.58和1.30 lg。可见,近海水源水是池塘环境中ARGs的主要来源,使用强氯精对其进行氧化消毒,可明显降低水体中常见ARGs水平,有助于防控ARGs在养殖环境中的传播。
  • 图  1  各样品中ARGs总浓度变化

    注:不同字母代表显著差异 (P<0.05)。

    Figure  1.  Total ARGs concentrations in samples

    Note: Different letters indicate significant difference (P<0.05).

    图  2  不同时刻相同采样点各种优势ARGs的浓度

    Figure  2.  Concentrations of various dominant ARGs at same sampling point at different time

    图  3  3次取样时ARGs总浓度对比

    Figure  3.  Comparison of total ARGs concentrations for three samplings

    表  1  qPCR 反应体系

    Table  1.   qPCR reaction solution

    qPCR反应试剂
    qPCR reaction solution
    各组分加样量
    Amount of usage/μL
    荧光定量染料 TB® Premix Ex Taq™ (Tli RNaseH Plus) (2×) 5
    前置引物 (50 μmol·L‒1) Forward primer (50 μmol·L‒1) 0.04
    后置引物 (50 μmol·L‒1) Reverse primer (50 μmol·L‒1) 0.04
    样品DNA Templatea 2
    超纯水 dd H2O 2.92
    体系总量 Total 10
    注:a. 每次定量均设置阳性对照和阴性对照,阳性对照为建立标曲所用标准质粒,阴性对照为dd H2O。 Note: a. Positive control and negative control were set for each run. The positive control was the standard plasmid used to establish the standard curve, and the negative control was dd H2O.
    下载: 导出CSV

    表  2  本研究中qPCR 所需引物

    Table  2.   Primers used for quantitative PCR in this study

    基因
    Gene
    引物对
    Primer pair
    序列 (5'—3')
    Sequence (5'−3')
    退火温度
    Annealing temperature/℃
    片段大小
    Amplicon size/bp
    参考文献
    Reference
    sul1 FW CGCACCGGAAACATCGCTGCAC 62 163 [20]
    RV TGAAGTTCCGCCGCAAGGCTCG
    sul2 FV TCCGGTGGAGGCCGGTATCTGG 62 191 [20]
    RV CGGGAATGCCATCTGCCTTGAG
    tetX FW AGCCTTACCAATGGGTGTAAA 55 280 [21]
    RV TTCTTACCTTGGACATCCCG
    cmlA FW GCCAGCAGTGCCGTTTAT 55 158 [22]
    RV GGCCACCTCCCAGTAGAA
    floR FW CGGTCGGTATTGTCTTCACG 56 171 [22]
    RV TCACGGGCCACGCTGTAT
    qnrA FW AGGATTTCTCACGCCAGGATT 57 124 [22]
    RV CCGCTTTCAATGAAACTGCA
    tetW FW GAGAGCCTGCTATATGCCAGC 55 168 [23]
    RV GGGCGTATCCACAATGTTAAC
    注:FW. 上游引物;RV. 下游引物。 Note: FW. Forward primer; RV. Reverse primer.
    下载: 导出CSV

    表  3  样品中环境因子检测结果

    Table  3.   Monitoring results of environmental factors in samples mg·L−1

    检测样品
    Sample
    固体悬浮物
    SS
    氨氮
    NH3-N
    硝酸盐氮
    NO3 -N
    亚硝酸盐氮
    NO2 -N
    无机氮
    IN
    总氮
    TN
    磷酸盐
    PO4 3−
    化学需氧量
    COD
    SY1 36 0.019 0.012 <0.003 0.034 1.18 0.15 22.2
    SY2 9 0.014 <0.006 <0.003 0.016 1.15 0.023 11.2
    SY3 10 0.025 <0.006 <0.003 0.025 0.67 0.017 9.2
    XSC1 2 0.037 <0.006 0.003 0.043 0.39 0.069 2.9
    XSC2 12 0.009 0.009 <0.003 0.019 0.43 0.063 3.2
    XSC3 9 <0.006 0.011 <0.003 0.015 0.75 0.074 4.9
    XD1 4 <0.006 0.25 <0.003 0.255 0.55 0.047 0.8
    XD2 2 0.008 0.311 <0.003 0.322 0.81 0.026 <0.5
    XD3 6 <0.006 0.009 <0.003 0.013 0.69 0.017 <0.5
    注:<. 检测结果小于检出限以,加粗数值代表检出限。 Note: <. Detection result is less than the detection limit, and value in bold represent the detection limit.
    下载: 导出CSV

    表  4  样品中ARGs的Ct值检测结果

    Table  4.   Ct value of ARGs in samples

    抗生素抗性基因
    ARG
    Ct值 Ct value
    样品
    Sample
    空白
    Blank
    sul1 19.24~25.18 29.33~29.85
    sul2 12.4~17.5 26.53~27.39
    floR 26.99~30.32 33.2~33.93
    cmlA 31.21~34.25 33.65~34.79
    tetw 29.02~31.8 30.21~30.48
    tetX 23.19~30.76 31.7~32.36
    qnrA 33.61~34.05
    下载: 导出CSV

    表  5  沉淀及氧化处理对水体中ARGs去除量比较

    Table  5.   Comparison of removal effects of ARGs in water by       sedimentation and oxidation treatment    lg

    处理 
    Treatment 
    总 ARGs
    Total ARGs
    floR sul1 sul2 tetX
    沉淀 Precipitation 0.86* 0.34 0.17 0.86* −0.32
    氧化 Oxidation 1.58* 0.71* 0.94* 1.58* 1.30*
    注:*. 表示处理后较水源水中ARGs浓度的差异性显著 (P<0.05)。 Note: *. There is a significant difference in ARGs concentrations in the treated water compared with that in the source water (P<0.05).
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-12-01
  • 修回日期:  2022-03-23
  • 录用日期:  2022-03-31
  • 网络出版日期:  2022-04-12
  • 刊出日期:  2022-12-05

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