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水体中致病菌富集和核酸提取方法及优化

吴妍 张晓 张良 邢方潇 张岚

吴妍, 张晓, 张良, 邢方潇, 张岚. 水体中致病菌富集和核酸提取方法及优化[J]. 中国公共卫生, 2023, 39(11): 1496-1500. doi: 10.11847/zgggws1141801
引用本文: 吴妍, 张晓, 张良, 邢方潇, 张岚. 水体中致病菌富集和核酸提取方法及优化[J]. 中国公共卫生, 2023, 39(11): 1496-1500. doi: 10.11847/zgggws1141801
WU Yan, ZHANG Xiao, ZHANG Liang, XING Fangxiao, ZHANG Lan. Membrane filtration enrichment and optimization of nucleic acid extraction for qPCR detection of pathogenic bacteria in water – an experiment with Mycobacterium abscessus tester strain[J]. Chinese Journal of Public Health, 2023, 39(11): 1496-1500. doi: 10.11847/zgggws1141801
Citation: WU Yan, ZHANG Xiao, ZHANG Liang, XING Fangxiao, ZHANG Lan. Membrane filtration enrichment and optimization of nucleic acid extraction for qPCR detection of pathogenic bacteria in water – an experiment with Mycobacterium abscessus tester strain[J]. Chinese Journal of Public Health, 2023, 39(11): 1496-1500. doi: 10.11847/zgggws1141801

水体中致病菌富集和核酸提取方法及优化

doi: 10.11847/zgggws1141801
基金项目: 国家重点研发计划(2021YFC3200805 – 5);国家重点研发计划(2022YFC3204702)
详细信息
    作者简介:

    吴妍(1998 – ),硕士,研究方向:水环境与健康

    通信作者:

    张岚,E-mail:zhanglan@nieh.chinacdc.cn

Membrane filtration enrichment and optimization of nucleic acid extraction for qPCR detection of pathogenic bacteria in water – an experiment with Mycobacterium abscessus tester strain

More Information
  • 摘要:   目的  建立一种灵敏、可靠、快速对水体中致病菌富集和核酸提取的前处理方法,用于水体致病菌荧光定量PCR检测。  方法  通过加标准菌株脓肿分枝杆菌、原菌液梯度稀释,采用离心柱法和磁珠法核酸提取试剂盒结合不同滤膜洗脱方式进行核酸提取;采用不同材质、尺寸、数量的击打物质及均质仪循环次数等对核酸提取条件进行优化;所得核酸进行荧光定量PCR检测,比较各方法的提取效率;采用最优提取条件,对加标水样进行荧光定量PCR检测,考察全流程回收率和检出限。  结果  采用0.20 μm滤膜过滤 + 7颗大锆珠均质仪循环6次 + TIANMicrobe Magnetic Envir-DNA Kit方法回收率最高(81.03% ± 19.23%),检出限达到6.20 CFU/100 mL。  结论  均质仪 + TIANMicrobe Magnetic Envir-DNA Kit法更适用于水体中致病菌的富集和核酸提取。
  • 图  1  9种核酸提取方法Ct值比较

    Figure  1.  qPCR Ct values for nucleic acid detection of Mycobacterium abscessus in water samples with 9 pre-treatment procedures

    图  2  采用最优前处理方法提核酸建立的标准曲线

    Figure  2.  Standard curve of qPCR Ct value against nucleic acid concentration logarithm established by using 10 times gradient dilution of Mycobacterium abscessus standard plasmid

    表  1  核酸提取条件优化

    Table  1.   qPCR Ct values for nucleic acid detection of Mycobacterium abscessus in water sampes under different extraction conditions: material, size and number of beads, homogenization cycles

    核酸提取条件次数Ct1Ct2$\bar{x}$
    击打物 大钢珠 28.212 28.011 28.112
    小钢珠 28.542 27.355 27.949
    大锆珠 27.103 27.679 27.391
    中锆珠 28.053 28.128 28.091
    小锆珠 27.743 28.412 28.078
    击打物大锆珠数量(颗) 3 3 31.771 31.961 31.866
    6 32.636 32.380 32.508
    5 3 28.346 27.472 27.909
    6 30.706 31.033 30.870
    7 3 25.961 26.370 26.166
    6 26.384 25.783 26.084
    9 3 26.283 26.505 26.394
    6 27.321 26.605 26.963
    循环次数(次) 3 27.034 27.831 27.433
    6 26.920 26.736 26.828
    9 27.373 26.745 27.059
    下载: 导出CSV

    表  2  最优方法组合处理加标水样的总回收率

    Table  2.   Total recoveries of Mycobacterium abscessus in spiked water samples using optimal pre-treatment: homogenization plus extraction with TIANMicrobe Magnetic Envir-DNA Kit

    加标菌液浓度
    (CFU/mL)
    加标水样检出率
    (%,n = 6)
    加标水样检测浓度
    (copies/μL,$\bar x \pm s$)
    总回收率
    (%,$\bar x \pm s$)
    3.1 × 102 83.33 2.31 ± 1.27 74.65 ± 41.11
    6.2 × 102 100.00 5.02 ± 1.19 81.03 ± 19.23
    下载: 导出CSV
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出版历程
  • 接收日期:  2023-03-21
  • 录用日期:  2023-09-22
  • 修回日期:  2023-06-07
  • 网络出版日期:  2023-11-28
  • 刊出日期:  2023-11-01

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