WO2021142988A1 - 一种利用古菌分子标记otu69快速检测城市搬迁地土壤全汞含量的方法 - Google Patents

一种利用古菌分子标记otu69快速检测城市搬迁地土壤全汞含量的方法 Download PDF

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WO2021142988A1
WO2021142988A1 PCT/CN2020/090168 CN2020090168W WO2021142988A1 WO 2021142988 A1 WO2021142988 A1 WO 2021142988A1 CN 2020090168 W CN2020090168 W CN 2020090168W WO 2021142988 A1 WO2021142988 A1 WO 2021142988A1
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soil
sequence
total mercury
mercury content
probe
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French (fr)
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张浪
韩继刚
张维维
金荷仙
王香春
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上海市园林科学规划研究院
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Definitions

  • the invention belongs to the field of biotechnology and relates to a method for rapidly detecting the total mercury content of soil in urban relocation sites by using archaeal molecular marker OTU69.
  • Mercury is one of the most harmful heavy metals to the human body, and it is one of the 129 priority pollutants.
  • the mercury in the soil mainly comes from: 1) soil parent material.
  • the mercury in the soil parent material is the most basic source.
  • the content of mercury in protoliths directly determines the content of mercury in the soil; 2) atmospheric deposition, mercury in the atmosphere After entering the soil, most of them are quickly adsorbed or fixed by the clay minerals and organic matter in the soil, and are enriched in the surface of the soil; 3) Direct pollution, mainly including the stacking of industrial production waste and municipal solid waste, and unreasonable application of mercury-containing fertilizers And pesticides and irrigation.
  • Mercury and its compounds have strong neurotoxicity and teratogenic effects, and pose a serious threat to the ecological environment and human health. The problem of soil mercury pollution has gradually received attention.
  • the purpose of the present invention is to provide a method for rapidly detecting the total mercury content of soil in urban relocation sites by using archaeal molecular marker OTU69.
  • the present invention also provides a method for detecting total mercury content in soil (Method A), which includes the following steps:
  • the amplification primer pair for real-time fluorescent quantitative PCR is the primer 524F-10-ext shown in sequence 4 of the sequence list and the primer Arch958R shown in sequence 5 of the sequence list -mod composition; the nucleotide sequence of the probe for real-time fluorescent quantitative PCR is shown in sequence 1 of the sequence list;
  • the Ct value is obtained by real-time fluorescent quantitative PCR; the copy number is calculated according to the Ct value, and the total mercury content in the soil sample is calculated by the copy number content in the soil sample.
  • the probe of real-time fluorescent quantitative PCR is Taqman probe.
  • the Taqman probe has a fluorescent group at the 5'end and a fluorescence quenching group at the 3'end.
  • the fluorescent group may specifically be FAM.
  • the fluorescence quenching group may specifically be TAMRA.
  • the copy number is the copy number of the target fragment of the probe.
  • the method for calculating the total mercury content in the soil sample by calculating the copy number content in the soil sample is: substituting the copy number content of the soil sample into a linear equation to obtain the total mercury content of the soil sample.
  • the method of calculating the copy number based on the Ct value is: Substituting the Ct value into the standard curve equation to obtain the copy number.
  • the preparation method of the standard curve equation is: connect the DNA molecule shown in sequence 2 of the sequence table to the pMD18-T vector to obtain OTU69 standard quality granules; use OTU69 standard quality granules to make the logarithm of the copy number as the independent variable and The standard curve equation with Ct as the dependent variable.
  • the logarithm of the copy number is the base 10 logarithm of the copy number.
  • Real-time PCR reaction system (20 ⁇ L): 10 ⁇ L Premix Ex Taq (Takara, Dalian, China), 0.4 ⁇ L primer 524F-10-ext, 0.4 ⁇ L primer Arch958R-mod, 0.2 ⁇ L probe, 2 ⁇ L template solution and 7 ⁇ L none Bacteria water.
  • concentration of primer 524F-10-ext is 0.2 ⁇ M
  • concentration of primer Arch958R-mod is 0.2 ⁇ M
  • concentration of probe is 0.1 ⁇ M.
  • the content of template DNA is 7 ng.
  • Real-time fluorescence quantitative PCR reaction program 95°C pre-denaturation 120s; 95°C denaturation 10s, 60°C annealing extension 45s, 45 cycles.
  • the present invention also provides a method for comparing the total mercury content of soil in different plots (Method B), which includes the following steps:
  • the present invention provides a DNA molecule (probe), as shown in sequence 1 of the sequence listing.
  • the DNA molecule may or may not be labeled with a label.
  • the label refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid.
  • Labels include, but are not limited to, dyes; radioactive labels, such as 32P; binding moieties, such as biotin (biotin); haptens, such as digoxin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or A fluorescent dye that can be combined with a part of the emission spectrum that can be suppressed or shifted by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetric measurement, X-ray diffraction or absorption, magnetism, enzyme activity, and the like.
  • the label may be a charged moiety (positive or negative) or alternatively, it may be neutral.
  • the label may include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the label is detectable. In some embodiments, the nucleic acid is directly detected without labeling (e.g., reading the sequence directly).
  • the present invention specifically provides a Taqman probe, the nucleotide sequence of which is shown in sequence 1 of the sequence list.
  • the Taqman probe has a fluorescent group at the 5'end and a fluorescence quenching group at the 3'end.
  • the fluorescent group may specifically be FAM.
  • the fluorescence quenching group may specifically be TAMRA.
  • the invention also protects the application of the DNA molecule or the Taqman probe in detecting or assisting in detecting the total mercury content of soil.
  • the invention also protects the application of the DNA molecule or the Taqman probe in comparing the total mercury content of the soil in different plots.
  • the present invention also protects the primer probe set, which is composed of a specific primer pair and a specific probe; the specific primer pair consists of primer 524F-10-ext shown in sequence 4 of the sequence list and primer Arch958R shown in sequence 5 of the sequence list. -mod composition; the nucleotide sequence of the specific probe is shown in sequence 1 of the sequence list.
  • the specific probe is a Taqman probe.
  • the Taqman probe has a fluorescent group at the 5'end and a fluorescence quenching group at the 3'end.
  • the fluorescent group may specifically be FAM.
  • the fluorescence quenching group may specifically be TAMRA.
  • the invention also protects the application of the primer probe set in detecting or assisting in detecting the total mercury content of the soil.
  • the invention also protects the application of the primer probe set in comparing the total mercury content of the soil in different plots.
  • the present invention also protects a kit, which includes the primer probe set.
  • the function of the kit is as follows (a) or (b):
  • the kit also includes a vector recording the method A or the method B.
  • any of the above-mentioned soils is green land soil.
  • any of the above-mentioned soils is China's green land soil.
  • any of the above-mentioned soils is urban green land soil.
  • any of the above-mentioned soils is the green land soil of the Yangtze River Delta in China.
  • any of the above-mentioned soils are urban green land soils in the Yangtze River Delta of China.
  • any of the above-mentioned soils is urban park green land soil.
  • any of the above-mentioned soils is the soil of the green land in the Yangtze River Delta of China.
  • Any of the above-mentioned land parcels is a park green land parcel in the Yangtze River Delta of China.
  • any of the above-mentioned soils is the soil of urban park green space in the Yangtze River Delta of China.
  • Any of the above-mentioned land parcels is an urban park green land parcel in the Yangtze River Delta of China.
  • any of the above-mentioned soils is the soil of the relocation site.
  • any of the above-mentioned soils is the soil of relocation sites in China.
  • Any of the land parcels mentioned above is a land parcel of relocation in China.
  • any of the above-mentioned soils is the soil of urban relocation sites.
  • any of the above-mentioned soils is the relocation soil of the Yangtze River Delta in China.
  • any of the above-mentioned soils is the soil of urban relocation sites in the Yangtze River Delta of China.
  • land parcels mentioned above are land parcels for urban relocation in the Yangtze River Delta of China.
  • the Yangtze River Delta is China's Shanghai, Jiangsu Province and Zhejiang province.
  • any of the above soil samples are taken from 0-20 cm surface soil.
  • Figure 1 shows the linear relationship between the copy number content of the target OTU and the total mercury content in the soil.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples, unless otherwise specified, are all conventional methods.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • Detection Indicator Detection method pH LY/T 1239-1999 Determination of the pH value of forest soil Conductivity LY/T 1251-1999 Forest soil water-soluble salt analysis (conductivity method) Organic matter NY/T 1121.6-2006 Determination of Soil Organic Matter Total nitrogen LY/T 1228-2015 Determination of forest soil nitrogen (Kjeldahl method) Available nitrogen LY/T 1228-2015 Determination of forest soil nitrogen (alkaline hydrolysis-diffusion method) Total phosphorus LY/T 1232-2015 Determination of phosphorus in forest soils (alkali fusion molybdenum antimony colorimetric method) Total potassium LY/T 1234-2015 Forest soil potassium determination (alkali melting method) Available phosphorus DB31/T 661-2012 Appendix F AB-DTPA Extraction/Inductively Coupled Plasma Mass Spectrometer Available potassium DB31/T 661-2012 Appendix F AB-DTPA extraction/inductively coupled plasma emission
  • Detection Indicator Detection method Total arsenic GB/T22105.2-2008 Soil quality Determination of total mercury, total arsenic and total lead by atomic fluorescence method all bronze Total digestion inductively coupled plasma mass spectrometer method Full zinc Total digestion inductively coupled plasma mass spectrometer method Full lead Total digestion inductively coupled plasma mass spectrometer method Full chromium Total digestion inductively coupled plasma mass spectrometer method Full nickel Same as the above method for total digestion inductively coupled plasma mass spectrometer Available calcium Refer to DB31/T 661-2012 Appendix F AB-DTPA extraction/inductively coupled plasma emission spectrometer Available manganese DB31/T 661-2012 Appendix F AB-DTPA extraction/inductively coupled plasma emission spectrometer Available zinc DB31/T 661-2012 Appendix F AB-DTPA extraction/inductively coupled plasma emission spectrometer Total mercury USEPA 7473-2007 Thermal Decomposition Homogenization Atomic Ab
  • the difference of effective calcium detection is only that the detection target is effective calcium.
  • Sampling method The collection of soil samples follows the principle of multi-point mixing. Eight sampling points are selected for each plot, and the surface soil of 0-20 cm is collected with a 2.5 cm diameter soil drill, and then mixed into 1 soil sample.
  • the soil samples are mixed evenly and passed through a 2mm sieve to remove plant roots, gravel and other debris. Then divide each soil sample into two. One sample was air-dried naturally, and then a sample was used to determine the chemical properties of the soil in step two; the other sample was stored at -80°C, and then sampled for use in the extraction of total soil DNA in step three.
  • step one the natural air-dried soil samples are analyzed and determined for soil quality indicators. See Table 2 for the determination results of soil quality indicators.
  • the DNA quality was detected by Nanodrop 2000 ultra-micro spectrophotometer and 0.8% agarose gel electrophoresis (5V cm -1 , 45 min).
  • the OD260/OD280 of the 76 DNA samples were in the range of 1.8-2.0, the maximum value of OD260/OD280 was 1.98, and the minimum value of OD260/OD280 was 1.81.
  • the DNA sample was used as a template, and a primer pair composed of primer 524F-10-ext and primer Arch958R-mod was used for PCR amplification.
  • a primer pair composed of primer 524F-10-ext and primer Arch958R-mod was used for PCR amplification.
  • the PCR products are subjected to 2% agarose gel electrophoresis, and then the target bands are cut and purified by the GeneJET Gel Recovery Kit (Thermo Scientific), and then the sequencing library is constructed using the Illumina MiSeq sequencing platform ( Illumina, San Diego, CA, USA) for sequencing.
  • Primer 524F-10-ext and primer Arch958R-mod are universal primers for archaea, and the target sequence is located in the variable region of archaeal 16S rRNA gene V4-V5.
  • 524F-10-ext (sequence 4 in the sequence listing): 5'-TGYCAGCCGCCGCGGTAA-3';
  • the reaction system for PCR amplification is 30 ⁇ L.
  • the active ingredient is 15 ⁇ L High-Fidelity PCR Master Mix (New England Biolabs), primers and template DNA.
  • the concentration of primer 524F-10-ext and primer Arch958R-mod are both 0.2 ⁇ M.
  • the content of template DNA is 10 ng.
  • PCR amplification reaction program 95°C pre-denaturation 3min; 95°C denaturation 30s, 55°C annealing 30s, 72°C extension 45s, 33 cycles; 72°C extension 5min.
  • Each sample selected 27765 archaeal 16S rRNA gene sequences for subsequent analysis.
  • the archaeal 16S rRNA gene sequences were classified with 95% sequence similarity, and a total of 580 OTUs were obtained.
  • the total mercury content of soil showed a significant negative correlation, with a correlation coefficient r of -0.557; the correlation coefficient between the abundance of archaea OTU384 in the soil and the total mercury content of the soil was -0.236.
  • the abundance of archaea OTU69 or OTU384 in the soil can be used to reflect the total mercury content in the soil.
  • Example 2 Establish a linear relationship between OTU and soil chemical characteristics
  • Probe69-1 probe (sequence 1 in the sequence listing): 5'-ACCCGCTCAACGGTTGGGCT-3'.
  • Probe69-2 probe (sequence 6 in the sequence listing): 5'-TGATGGGATGGCCTCGAGCT-3'.
  • Probe69-1 is a Taqman probe with a fluorescent group FAM at the 5'end and a fluorescence quenching group TAMRA at the 3'end.
  • Probe69-2 is a Taqman probe with a fluorescent group FAM at the 5'end and a fluorescence quenching group TAMRA at the 3'end.
  • Probe384-1 probe (sequence 7 in the sequence listing): 5'-TGAACAGGCTTAGTGCCTATT-3'.
  • Probe384-2 probe (sequence 8 of the sequence listing): 5'-AGTGCCTATTCAGTGCCGCA-3'.
  • Probe384-1 is a Taqman probe with a fluorescent group FAM at the 5'end and a fluorescence quenching group TAMRA at the 3'end.
  • Probe384-2 is a Taqman probe with a fluorescent group FAM at the 5'end and a fluorescence quenching group TAMRA at the 3'end.
  • a soil sample with a gradient distribution of total mercury content was randomly selected from the soil samples in step 1 of Example 1.
  • the extraction method of total DNA is the same as that of step 3 in Example 1.
  • step 2 Take the template solution (that is, the total DNA solution obtained in step 1), and use 96 real-time fluorescent quantitative PCR instrument for fluorescent quantitative PCR (probe method).
  • a primer pair consisting of primer 524F-10-ext and primer Arch958R-mod is used. Use Probe69-1 probe or Probe69-2 probe or Probe384-1 probe or Probe384-2 probe.
  • Reaction system (20 ⁇ L): 10 ⁇ L Premix Ex Taq (Takara, Dalian, China), 0.4 ⁇ L primer 524F-10-ext, 0.4 ⁇ L primer Arch958R-mod, 0.2 ⁇ L probe, 2 ⁇ L template solution and 7 ⁇ L sterile water.
  • concentration of primer 524F-10-ext is 0.2 ⁇ M
  • concentration of primer Arch958R-mod is 0.2 ⁇ M
  • concentration of probe is 0.1 ⁇ M.
  • the content of template DNA is 7 ng.
  • Reaction program 95°C pre-denaturation for 120s; 95°C denaturation for 10s, 60°C annealing extension for 45s, 45 cycles.
  • the specificity of amplification is determined by the melting curve.
  • the DNA molecule (amplified from total soil DNA) shown in sequence 2 of the sequence listing was ligated with the pMD18-T vector to obtain OTU69 standard quality particles.
  • Using TE buffer as the solvent prepare standard quality pellet solutions containing different concentrations of OTU69 standard quality pellets (measure the DNA concentration in the standard quality pellet solution with a Nanodrop 2000 ultra-micro spectrophotometer, and convert the DNA copy number, which is the OTU69 copy number) ;
  • the DNA molecule (amplified from total soil DNA) shown in sequence 3 of the sequence listing was ligated with the pMD18-T vector to obtain OTU384 standard quality pellets.
  • Using TE buffer as the solvent prepare standard quality pellet solutions containing different concentrations of OTU384 standard quality pellets (measure the DNA concentration in the standard quality pellet solution with a Nanodrop2000 ultra-micro spectrophotometer, and convert the DNA copy number, which is the OTU384 copy number);
  • the standard curve equation with the independent variable and Ct as the dependent variable.
  • the efficiency of fluorescence quantitative PCR amplification is between 90% and 110%.
  • the results of the measured total mercury value (unit: mg/kg), Ct value and the target OTU copy number content (unit: copy number/g) of the soil sample are shown in Table 3.
  • the measured value of total mercury in the soil sample is the data obtained in step 2 of Example 1.
  • the Ct value and the copy number content of the target OTU in the soil sample are the data obtained in step 2 of this embodiment.
  • Probe69-1 probe to perform fluorescence quantitative PCR, the relative abundance of the target OTU in the soil (reflected in the copy number of the target OTU in the soil) has a good linear relationship with the total mercury content in the soil.
  • Soil sample 1 collected from park green spaces in Songjiang District; soil sample 2, collected from park green spaces in Jinshan District; soil sample 3, collected from park green spaces in Huangpu District; soil sample 4, collected from park green spaces in Jing’an District; Soil sample 5 was collected from park green spaces in Putuo District; soil sample 6 was collected from park green spaces in Minhang District.
  • Example 3 Molecular detection of total mercury content in soil samples of unknown urban green spaces
  • Sampling method The collection of soil samples follows the principle of multi-point mixing. Eight sampling points are selected for each plot, and the surface soil of 0-20 cm is collected with a 2.5 cm diameter soil drill, and then mixed into 1 soil sample.
  • the soil samples are mixed evenly and passed through a 2mm sieve to remove plant roots, gravel and other debris. Then divide each soil sample into two. One sample was air-dried naturally, and then a sample was taken to detect the total mercury content to obtain the measured value of the total mercury content (unit: mg/kg); the other sample was stored at -80°C and then sampled for total DNA extraction from the soil.
  • Reaction system (20 ⁇ L): 10 ⁇ L Premix Ex Taq (Takara, Dalian, China), 0.4 ⁇ L primer 524F-10-ext, 0.4 ⁇ L primer Arch958R-mod, 0.2 ⁇ L Probe69-1 probe, 2 ⁇ L template solution and 7 ⁇ L sterile water .
  • concentration of primer 524F-10-ext is 0.2 ⁇ M
  • concentration of primer Arch958R-mod is 0.2 ⁇ M
  • concentration of Probe69-1 probe is 0.1 ⁇ M.
  • the content of template DNA is 7 ng.
  • Reaction program 95°C pre-denaturation for 120s; 95°C denaturation for 10s, 60°C annealing extension for 45s, 45 cycles.
  • step 3 of the second embodiment For the preparation method of the standard curve equation, refer to step 3 of the second embodiment.
  • Example 4 Molecular detection of total mercury content in soil samples from relocation sites of unknown cities
  • the invention discloses a method for detecting the total mercury content of the soil or comparing the total mercury content of the soil in different plots, which has the following functions: detecting the total mercury content of the soil or comparing the total mercury content of the soil in different plots, the sample demand is small, and no pre-treatment is required , The required time is short, the labor cost is low, and the rapid automatic detection of large quantities of samples can be realized.
  • the invention has important application and promotion value for the evaluation of soil samples.

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Abstract

提供了一种利用古菌分子标记OTU69快速检测城市搬迁地土壤全汞含量的方法。本发明提供了一种DNA分子（探针），如序列表的序列1所示。还提供了所述探针在检测或辅助检测土壤全汞含量中的应用和所述探针在比较不同地块的土壤全汞含量中的应用。

Description

一种利用古菌分子标记OTU69快速检测城市搬迁地土壤全汞含量的方法 技术领域
本发明属于生物技术领域，涉及一种利用古菌分子标记OTU69快速检测城市搬迁地土壤全汞含量的方法。
背景技术
随着城市化的快速发展，目前我国城市，尤其是特大城市，已进入“城市再开发”为主导的城市发展阶段，城市搬迁地大量出现，在东部沿海经济发达地区的城市尤其明显。以上海为例，建成区范围内80％-90％的建设绿地都是在城中村拆迁、旧工厂搬迁等地块上开展的，对其土壤质量进行科学客观的监测和评价，是进行城市搬迁地生态修复和园林绿化的前提和重要参考依据。
目前对城市搬迁地土壤质量指标的检测主要是沿用传统的理化检测方法，样品需求量大，样品前处理过程复杂、周期长，尤其所需要的人力资源成本较高，难以实现对大批量样品的快速检测。土壤微生物对土壤环境的变化具有综合性、敏感性和功能性的特点，探索通过检测特定微生物类群的丰度来实现对城市搬迁地土壤质量指标的快速和自动化检测技术具有重要的现实意义。
汞元素是对人体毒害最大的重金属之一，属于129种优先控制污染物之一。土壤中的汞主要来源于：1)土壤母质，土壤母质中的汞是最基本的来源，原生岩中汞元素的含量，直接决定着土壤中汞的含量；2)大气沉降，大气中的汞进入土壤后，绝大部分迅速被土壤中粘土矿物和有机物吸附或固定，富集于土壤表层；3)直接污染，主要包括工业生产废料和城市生活垃圾的堆放，不合理地施用含汞的肥料和农药以及灌溉等。汞及其化合物具有很强的神经毒性和致畸作用，对生态环境和人类健康构成严重威胁，土壤汞污染问题已逐步受到重视。
发明公开
本本发明的目的是提供一种利用古菌分子标记OTU69快速检测城市搬迁地土壤全汞含量的方法。
本发明还提供了一种检测土壤全汞含量的方法(方法甲)，包括如下 步骤：
以土壤样品的总DNA为模板，进行实时荧光定量PCR；实时荧光定量PCR的扩增引物对由序列表的序列4所示的引物524F-10-ext和序列表的序列5所示的引物Arch958R-mod组成；实时荧光定量PCR的探针的核苷酸序列如序列表的序列1所示；
实时荧光定量PCR获得Ct值；根据Ct值计算拷贝数，通过土壤样品中的拷贝数含量计算得到土壤样品中的全汞含量。
实时荧光定量PCR的探针为Taqman探针。所述Taqman探针的5’末端具有荧光基团，3’末端具有荧光淬灭基团。所述荧光基团具体可为FAM。所述荧光淬灭基团具体可为TAMRA。
所述拷贝数为所述探针的靶标片段的拷贝数。
通过土壤样品中的拷贝数含量计算得到土壤样品中的全汞含量的实现方法为：将土壤样品的拷贝数含量代入线性方程，得到土壤样品的全汞含量。
线性方程为：y＝-0.1811x+0.6508；y代表全汞含量，单位为mg/kg；x代表拷贝数含量，单位为×10 7拷贝数/g。线性方程的R 2＝0.959。
根据Ct值计算拷贝数的方法为：将Ct值代入标准曲线方程，得到拷贝数。
所述标准曲线方程的制备方法为：将序列表的序列2所示的DNA分子与pMD18-T载体连接，得到OTU69标准品质粒；采用OTU69标准品质粒制作以拷贝数的对数为自变量且以Ct值为因变量的标准曲线方程。拷贝数的对数为拷贝数以10为底的对数。
土壤样品的总DNA的制备方法：采用MoBio
Figure PCTCN2020090168-appb-000001
DNA提取试剂盒(MoBio Laboratories,Carlsbad,Inc.,CA,USA)提取土壤样品的总DNA。
实时荧光定量PCR具体采用
Figure PCTCN2020090168-appb-000002
96实时荧光定量PCR仪。
实时荧光定量PCR的反应体系(20μL)：10μL Premix Ex Taq(Takara,Dalian,China)、0.4μL引物524F-10-ext、0.4μL引物Arch958R-mod、0.2μL探针、2μL模板溶液和7μL无菌水。反应体系中，引物524F-10-ext的浓度为0.2μM，引物Arch958R-mod的浓度为0.2μM，探 针的浓度为0.1μM。反应体系中，模板DNA的含量为7ng。
实时荧光定量PCR的反应程序：95℃预变性120s；95℃变性10s、60℃退火延伸45s，45个循环。
本发明还提供了一种比较不同地块的土壤全汞含量的方法(方法乙)，包括如下步骤：
将两个以上地块的土壤样品分别按照方法甲进行检测；
根据检测结果比较各个地块的土壤全汞含量。
本发明提供了一种DNA分子(探针)，如序列表的序列1所示。
所述DNA分子(探针)可被标记物标记也可不被标记物标记。所述标记物指可用于提供可检测的效果且可以连接至核酸的任何原子或分子。标记物包括但不限于染料；放射性标记，诸如32P；结合部分，诸如生物素(biotin)；半抗原，诸如地高辛(DIG)；发光、发磷光或发荧光部分；和单独的荧光染料或与可以通过荧光共振能量转移(FRET)抑制或移动发射光谱的部分组合的荧光染料。标记可以提供可通过荧光、放射性、比色、重量测定、X射线衍射或吸收、磁性、酶活性等检测的信号。标记可以是带电荷的部分(正电荷或负电荷)或可选地，可以是电荷中性的。标记可以包括核酸或蛋白序列或由其组合，只要包含标记的序列是可检测的。在一些实施方案中，核酸在没有标记的情况下直接检测(例如，直接读取序列)。
本发明具体提供了一种Taqman探针，其核苷酸序列如序列表的序列1所示。所述Taqman探针的5’末端具有荧光基团，3’末端具有荧光淬灭基团。所述荧光基团具体可为FAM。所述荧光淬灭基团具体可为TAMRA。
本发明还保护所述DNA分子或所述Taqman探针在检测或辅助检测土壤全汞含量中的应用。
本发明还保护所述DNA分子或所述Taqman探针在比较不同地块的土壤全汞含量中的应用。
本发明还保护引物探针组，由特异引物对和特异探针组成；所述特异引物对由序列表的序列4所示的引物524F-10-ext和序列表的序列5所示的引物Arch958R-mod组成；所述特异探针的核苷酸序列如序列表的序列1所示。所述特异探针为Taqman探针。所述Taqman探针的5’末端具有荧 光基团，3’末端具有荧光淬灭基团。所述荧光基团具体可为FAM。所述荧光淬灭基团具体可为TAMRA。
本发明还保护所述引物探针组在检测或辅助检测土壤全汞含量中的应用。
本发明还保护所述引物探针组在比较不同地块的土壤全汞含量中的应用。
本发明还保护一种试剂盒，包括所述引物探针组。
所述试剂盒的功能为如下(a)或(b)：
(a)检测或辅助检测土壤全汞含量；
(b)比较不同地块的土壤全汞含量。
所述试剂盒还包括记载有所述方法甲或所述方法乙的载体。
以上任一所述土壤为绿地土壤。
以上任一所述地块为绿地地块。
以上任一所述土壤为中国绿地土壤。
以上任一所述地块为中国绿地地块。
以上任一所述土壤为城市绿地土壤。
以上任一所述地块为城市绿地地块。
以上任一所述土壤为中国长江三角洲的绿地土壤。
以上任一所述地块为中国长江三角洲的绿地地块。
以上任一所述土壤为中国长江三角洲的城市绿地土壤。
以上任一所述地块为中国长江三角洲的城市绿地地块。
以上任一所述土壤为公园绿地土壤。
以上任一所述地块为公园绿地地块。
以上任一所述土壤为城市公园绿地土壤。
以上任一所述地块为城市公园绿地地块。
以上任一所述土壤为中国长江三角洲的公园绿地土壤。
以上任一所述地块为中国长江三角洲的公园绿地地块。
以上任一所述土壤为中国长江三角洲的城市公园绿地土壤。
以上任一所述地块为中国长江三角洲的城市公园绿地地块。
以上任一所述土壤为搬迁地土壤。
以上任一所述地块为搬迁地地块。
以上任一所述土壤为中国搬迁地土壤。
以上任一所述地块为中国搬迁地地块。
以上任一所述土壤为城市搬迁地土壤。
以上任一所述地块为城市搬迁地地块。
以上任一所述土壤为中国长江三角洲的搬迁地土壤。
以上任一所述地块为中国长江三角洲的搬迁地地块。
以上任一所述土壤为中国长江三角洲的城市搬迁地土壤。
以上任一所述地块为中国长江三角洲的城市搬迁地地块。
长江三角洲为中国的上海市、江苏省和浙江省。
以上任一所述土壤样品取自0-20cm表层土壤。
附图说明
图1为目标OTU的拷贝数含量与土壤全汞含量的线性关系。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
土壤质量指标的测定依据相关国家标准、行业标准和地方标准进行，详见表1。
表1土壤质量指标测定方法
检测指标 检测方法
pH LY/T 1239-1999森林土壤pH值的测定
电导率 LY/T 1251-1999森林土壤水溶性盐分分析(电导法)
有机质 NY/T 1121.6-2006土壤有机质的测定
全氮 LY/T 1228-2015森林土壤氮的测定(凯氏定氮法)
有效氮 LY/T 1228-2015森林土壤氮的测定(碱解-扩散法)
全磷 LY/T 1232-2015森林土壤磷的测定(碱熔钼锑抗比色法)
全钾 LY/T 1234-2015森林土壤钾的测定(碱熔法)
有效磷 DB31/T 661-2012附录F AB-DTPA浸提/电感耦合等离子体质谱仪
有效钾 DB31/T 661-2012附录F AB-DTPA浸提/电感耦合等离子体发射光谱仪
有效硫 DB31/T 661-2012附录F AB-DTPA浸提/电感耦合等离子体质谱仪
有效氯 参考DB31/T 661-2012中附录E(水饱和浸提)
交换性钠 参考DB31/T 661-2012中附录E(水饱和浸提)
检测指标 检测方法
全砷 GB/T22105.2-2008土壤质量总汞、总砷、总铅的测定原子荧光法
全铜 全消解电感耦合等离子体质谱仪方法
全锌 全消解电感耦合等离子体质谱仪方法
全铅 全消解电感耦合等离子体质谱仪方法
全铬 全消解电感耦合等离子体质谱仪方法
全镍 同上全消解电感耦合等离子体质谱仪方法
有效钙 参照DB31/T 661-2012附录F AB-DTPA浸提/电感耦合等离子体发射光谱仪
有效锰 DB31/T 661-2012附录F AB-DTPA浸提/电感耦合等离子体发射光谱仪
有效锌 DB31/T 661-2012附录F AB-DTPA浸提/电感耦合等离子体发射光谱仪
全汞 USEPA 7473-2007热分解齐化原子吸收光度法
注：与所参照的方法相比，有效钙检测的区别点仅在于检测目标物为有效钙。
实施例1、发现与土壤全汞含量相关的OUT
一、土壤样品的采集
2017年11月，在中国上海市16个行政区的代表性公园绿地设置研究样地。
采样方法：土壤样品采集遵循多点混合的采样原则，每块样地选择8个取样点，利用直径2.5cm土钻分别采集0-20cm表层土壤，之后混合成1个土壤样品。
共采集土壤样品76个。
土壤样品混合均匀，过2mm筛，去除植物根系、碎石等杂物。然后将每个土壤样品分为两份。一份样品自然风干，然后取样用于步骤二中测定土壤化学性质；另一份样品于-80℃保存，然后取样用于步骤三中土壤总DNA提取。
二、土壤质量指标的分析测定
步骤一中自然风干的土壤样品，进行土壤质量指标的分析测定。土壤质量指标的测定结果见表2。
表2土壤质量指标检测结果
检测指标 最小值 最大值 平均值
pH 5.32 8.79 7.92
电导率(μS/cm) 60.70 656.52 142.57
有机质(g/kg) 7.10 46.62 27.59
检测指标 最小值 最大值 平均值
全氮(g/kg) 0.47 2.34 1.13
有效氮(mg/kg) 25.85 152.64 84.68
全磷(g/kg) 0.42 1.00 0.68
全钾(g/kg) 15.80 25.43 19.08
有效磷(mg/kg) 0.85 48.20 8.66
有效钾(mg/kg) 28.20 397.89 188.90
有效硫(mg/kg) 13.06 96.65 52.36
有效氯(mg/L) 4.16 1900.00 41.46
交换性钠(mg/L) 3.30 993.00 21.60
全砷(mg/kg) 4.81 13.50 8.73
全铜(mg/kg) 16.38 99.84 36.05
全锌(mg/kg) 87.38 223.87 125.52
全铅(mg/kg) 18.24 52.18 29.01
全铬(mg/kg) 55.40 101.00 72.90
全镍(mg/kg) 27.40 44.59 36.62
有效钙(mg/kg) 198.36 366.95 282.89
有效锰(mg/kg) 8.55 29.46 16.76
有效锌(mg/kg) 1.81 29.58 9.05
全汞(mg/kg) 0.05 0.57 0.21
三、土壤古菌种群多样性分析
1、步骤一中-80℃保存的土壤样品，提取总DNA。
提取土壤样品的总DNA，采用MoBio
Figure PCTCN2020090168-appb-000003
DNA提取试剂盒(MoBio Laboratories,Carlsbad,Inc.,CA,USA)。每个土壤样品重复提取两次，将两次提取的总DNA混合，得到DNA样本。76个土壤样品，得到相应的76个DNA样本。所有DNA样本均-80℃保存。
利用Nanodrop 2000超微量分光光度计和0.8％琼脂糖凝胶电泳(5V cm -1，45min)检测DNA质量。76个DNA样本OD260/OD280均在1.8-2.0范围之间，OD260/OD280最大值为1.98，OD260/OD280最小值为1.81。
2、古菌16S rRNA基因扩增及高通量测序
以DNA样本为模板，采用引物524F-10-ext和引物Arch958R-mod组成的引物对进行PCR扩增。完成PCR扩增后，将PCR产物进行2％琼脂糖凝胶电泳，然后利用GeneJET凝胶回收试剂盒(Thermo Scientific)对目的条带进行切胶纯化，然后构建测序文库，采用Illumina MiSeq测序平台(Illumina,San Diego,CA,USA)进行测序。
引物524F-10-ext和引物Arch958R-mod为古菌通用引物，靶序列位 于古菌16S rRNA基因V4-V5可变区。
524F-10-ext(序列表的序列4)：5'-TGYCAGCCGCCGCGGTAA-3'；
Arch958R-mod(序列表的序列5)：5'-YCCGGCGTTGAVTCCAATT-3'；
Y代表C或T；V代表G、A或C。
PCR扩增的反应体系为30μL。有效成分为15μL
Figure PCTCN2020090168-appb-000004
High-Fidelity PCR Master Mix(New England Biolabs)、引物和模板DNA。反应体系中，引物524F-10-ext和引物Arch958R-mod的浓度均为0.2μM。反应体系中，模板DNA的含量为10ng。
PCR扩增的反应程序：95℃预变性3min；95℃变性30s、55℃退火30s、72℃延伸45s，33个循环；72℃延伸5min。
3、高通量数据分析及结果
高通量测序结果生物信息学分析具体步骤如下：1)根据样品特异标签从原始数据中对同一样品序列进行抽提，形成单独的文件，并去除标签及引物序列；2)利用FLASH(V1.2.7)软件进行序列拼接；3)利用Qiime(V1.7.0)软件对测序完成后的原始序列进行质量过滤；4)利用UCHIME软件检测嵌合体，并将之删除；5)利用Uparse(v7.0.1001)软件在95％的相似性水平上划分操作分类单元(OTUs)；6)由于单拷贝序列的可靠性受到质疑，因此在后续分析中去除单拷贝序列；7)为了去除样品间不同测序深度的影响，样品OTU表格均一化到相同的测序深度；8)基于RDP数据库对OTUs进行序列比对，并确定其分类地位。
每个样本选取27765条古菌16S rRNA基因序列用于后续分析。在95％的序列相似度下对古菌16S rRNA基因序列进行归类，共获得580个OTUs。
四、土壤古菌类群与土壤质量指标的相关性分析
将步骤三得到的580个OTUs分别与步骤二得到的各个土壤化学指标数据进行Pearson相关性分析。
结果表明：580个OTUs中，土壤中古菌OTU69丰度与土壤全汞含量的相关性最强，土壤中古菌OTU384丰度与土壤全汞含量的相关性排第二；土壤中古菌OTU69丰度与土壤全汞含量呈显著负相关，相关系数r为-0.557；土壤中古菌OTU384丰度与土壤全汞含量的相关系数为-0.236。可利用检测土壤中古菌OTU69或古菌OTU384的丰度来反映土壤中全汞的 含量。
实施例2、建立OTU与土壤化学特征间的线性关系
一、古菌Marker基因探针设计
根据测序结果，设计用于检测OTU69的探针。
Probe69-1探针(序列表的序列1)：5'-ACCCGCTCAACGGTTGGGCT-3'。
Probe69-2探针(序列表的序列6)：5'-TGATGGGATGGCCTCGAGCT-3'。
Probe69-1探针为Taqman探针，5’末端具有荧光基团FAM，3’末端具有荧光淬灭基团TAMRA。Probe69-2探针为Taqman探针，5’末端具有荧光基团FAM，3’末端具有荧光淬灭基团TAMRA。
根据测序结果，设计用于检测OTU384的探针。
Probe384-1探针(序列表的序列7)：5'-TGAACAGGCTTAGTGCCTATT-3'。
Probe384-2探针(序列表的序列8)：5'-AGTGCCTATTCAGTGCCGCA-3'。
Probe384-1探针为Taqman探针，5’末端具有荧光基团FAM，3’末端具有荧光淬灭基团TAMRA。Probe384-2探针为Taqman探针，5’末端具有荧光基团FAM，3’末端具有荧光淬灭基团TAMRA。
二、土壤中古菌Marker基因拷贝数的测定
根据实测值，从实施例1的步骤一的土壤样品中随机选择全汞含量呈梯度分布的土壤样品。
1、取土壤样品，提取总DNA。
总DNA的提取方法同实施例1的步骤三的1。
2、取模板溶液(即步骤1得到的总DNA溶液)，利用
Figure PCTCN2020090168-appb-000005
96实时荧光定量PCR仪进行荧光定量PCR(探针法)。
采用引物524F-10-ext和引物Arch958R-mod组成的引物对。采用Probe69-1探针或Probe69-2探针或Probe384-1探针或Probe384-2探针。
反应体系(20μL)：10μL Premix Ex Taq(Takara,Dalian,China)、0.4μL引物524F-10-ext、0.4μL引物Arch958R-mod、0.2μL探针、2μL模板溶液和7μL无菌水。反应体系中，引物524F-10-ext的浓度为0.2μM，引物Arch958R-mod的浓度为0.2μM，探针的浓度为0.1μM。反应体系中，模板DNA的含量为7ng。
反应程序：95℃预变性120s；95℃变性10s、60℃退火延伸45s，45个循环。
通过溶解曲线确定扩增特异性。
将Ct值代入标准曲线方程，得到目标OTU的拷贝数，再计算得到土壤样品中目标OTU的拷贝数含量(单位为拷贝数/g，即每g干重的土壤样品中目标OTU的拷贝数)。
标准曲线方程的制作方法见步骤3。
3、制作荧光定量PCR标准曲线
将序列表的序列2所示的DNA分子(从土壤总DNA中扩增得到的)与pMD18-T载体连接，得到OTU69标准品质粒。采用TE缓冲液作为溶剂，制备含有不同浓度OTU69标准品质粒的标准品质粒溶液(通过Nanodrop 2000超微量分光光度计测定标准品质粒溶液中的DNA浓度，换算DNA拷贝数，即为OTU69拷贝数)；将各个标准品质粒溶液分别作为模板溶液，按照步骤2的方法进行检测(采用Probe69-1探针或Probe69-2探针)，获得以OTU69拷贝数的对数(以10为底的对数)为自变量且以Ct值为因变量的标准曲线方程。
序列2、
Figure PCTCN2020090168-appb-000006
将序列表的序列3所示的DNA分子(从土壤总DNA中扩增得到的)与pMD18-T载体连接，得到OTU384标准品质粒。采用TE缓冲液作为溶剂，制备含有不同浓度OTU384标准品质粒的标准品质粒溶液(通过Nanodrop2000超微量分光光度计测定标准品质粒溶液中的DNA浓度，换算DNA拷贝数，即为OTU384拷贝数)；将各个标准品质粒溶液分别作为模板溶液， 按照步骤2的方法进行检测(采用Probe384-1探针或Probe384-2探针)，获得以OTU384拷贝数的对数(以10为底的对数)为自变量且以Ct值为因变量的标准曲线方程。
序列3、
Figure PCTCN2020090168-appb-000007
荧光定量PCR扩增有效性在90％和110％之间。
三、古菌Marker基因拷贝数与土壤化学特征间线性关系的建立
土壤样品的全汞实测值(单位为mg/kg)、Ct值和土壤样品目标OTU的拷贝数含量(单位为拷贝数/g)的结果见表3。土壤样品的全汞实测值为实施例1的步骤二中获得的数据。Ct值和土壤样品中目标OTU的拷贝数含量为本实施例的步骤二获得的数据。利用Probe69-1探针进行荧光定量PCR，土壤中目标OTU相对丰度(体现为土壤中目标OTU拷贝数)与土壤全汞含量具有良好的线性关系。
表3
Figure PCTCN2020090168-appb-000008
注：土壤样品1，采集自松江区的公园绿地；土壤样品2，采集自金山区的公园绿地；土壤样品3，采集自黄浦区的公园绿地；土壤样品4，采集自静安区的公园绿地；土壤样品5，采集自普陀区的公园绿地；土壤样品6，采集自闵行区的公园绿地。
利用Probe69-1探针进行荧光定量PCR，土壤中目标OTU的拷贝数含量与土壤全汞含量的线性关系见图1。线性方程为：y＝-0.1811x+0.6508；R 2＝0.959；y代表全汞含量(mg/kg)，x代表目标OUT的拷贝数含量(×10 7拷贝数/g)。
实施例3、未知城市绿地土壤样品全汞含量的分子检测
在中国的上海、南京、苏州分别随机选择3块城市公园绿地。
采样方法：土壤样品采集遵循多点混合的采样原则，每块样地选择8个取样点，利用直径2.5cm土钻分别采集0-20cm表层土壤，之后混合成1个土壤样品。
土壤样品混合均匀，过2mm筛，去除植物根系、碎石等杂物。然后将每个土壤样品分为两份。一份样品自然风干，然后取样检测全汞含量，获得全汞含量实测值(单位为mg/kg)；另一份样品于-80℃保存，然后取样用于土壤总DNA提取。
1、提取土壤样品的总DNA。采用MoBio
Figure PCTCN2020090168-appb-000009
DNA提取试剂盒(MoBio Laboratories,Carlsbad,Inc.,CA,USA)。每个土壤样品重复提取两次，将两次提取的总DNA混合，得到DNA样本。
2、取模板溶液(即步骤1得到的DNA样本)，利用
Figure PCTCN2020090168-appb-000010
96实时荧光定量PCR仪进行荧光定量PCR(探针法)。
反应体系(20μL)：10μL Premix Ex Taq(Takara,Dalian,China)、0.4μL引物524F-10-ext、0.4μL引物Arch958R-mod、0.2μL Probe69-1探针、2μL模板溶液和7μL无菌水。反应体系中，引物524F-10-ext的浓度为0.2μM，引物Arch958R-mod的浓度为0.2μM，Probe69-1探针的浓度为0.1μM。反应体系中，模板DNA的含量为7ng。
反应程序：95℃预变性120s；95℃变性10s、60℃退火延伸45s，45个循环。
将Ct值代入标准曲线方程，得到OTU69的拷贝数，再计算得到土壤样品中OTU69的拷贝数含量(单位为拷贝数/g，即每g干重的土壤样品中OTU69的拷贝数)。
标准曲线方程的制作方法见实施例2的步骤二的3。
3、将土壤样品中OTU69的拷贝数含量代入线性方程，得到土壤样品的全汞含量的计算值(单位为mg/kg)。
线性方程为：y＝-0.1811x+0.6508；R 2＝0.959；y代表全汞含量(mg/kg)，x代表OTU69的拷贝数含量(×10 7拷贝数/g)。
土壤样品中OTU69的拷贝数含量(单位为拷贝数/g)、土壤样品的全汞含量的计算值(单位为mg/kg)、土壤样品的全汞含量的实测值(单位为mg/kg)的结果见表4。
表4
Figure PCTCN2020090168-appb-000011
实施例4、未知城市搬迁地土壤样品全汞含量的分子检测
在中国的上海、南京、苏州分别随机选择3块城市搬迁地。
方法同实施例3。
土壤样品中OTU69的拷贝数含量(单位为拷贝数/g)、土壤样品的全汞含量的计算值(单位为mg/kg)、土壤样品的全汞含量的实测值(单位为mg/kg)的结果见表5。
表5
Figure PCTCN2020090168-appb-000012
Figure PCTCN2020090168-appb-000013
工业应用
本发明公开了检测土壤全汞含量或比较不同地块的土壤全汞含量的方法，具有如下作用：检测土壤全汞含量或比较不同地块的土壤全汞含量，样品需求量小，无需前处理，所需时间短，人力成本低，可以实现对大批量样品的快速自动化检测。本发明对于土壤样品的测评具有重要应用推广价值。

Claims (12)

  1. 一种检测土壤全汞含量的方法，包括如下步骤：
    以土壤样品的总DNA为模板，进行实时荧光定量PCR；实时荧光定量PCR的扩增引物对由序列表的序列4所示的引物524F-10-ext和序列表的序列5所示的引物Arch958R-mod组成；实时荧光定量PCR的探针的核苷酸序列如序列表的序列1所示；
    实时荧光定量PCR获得Ct值；根据Ct值计算拷贝数，通过土壤样品中的拷贝数含量计算得到土壤样品中的全汞含量。
  2. 一种比较不同地块的土壤全汞含量的方法，包括如下步骤：
    将两个以上地块的土壤样品分别按照权利要求1的方法进行检测；
    根据检测结果比较各个地块的土壤全汞含量。
  3. 一种DNA分子，如序列表的序列1所示。
  4. 一种Taqman探针，其核苷酸序列如序列表的序列1所示。
  5. 权利要求3所述DNA分子在检测或辅助检测土壤全汞含量中的应用。
  6. 权利要求3所述DNA分子在比较不同地块的土壤全汞含量中的应用。
  7. 权利要求4所述Taqman探针在检测或辅助检测土壤全汞含量中的应用。
  8. 权利要求4所述Taqman探针在比较不同地块的土壤全汞含量中的应用。
  9. 引物探针组，由特异引物对和特异探针组成；所述特异引物对由序列表的序列4所示的引物524F-10-ext和序列表的序列5所示的引物Arch958R-mod组成；所述特异探针的核苷酸序列如序列表的序列1所示。
  10. 权利要求9所述引物探针组在检测或辅助检测土壤全汞含量中的应用。
  11. 权利要求9所述引物探针组在比较不同地块的土壤全汞含量中的应用。
  12. 一种试剂盒，包括权利要求9所述引物探针组。
PCT/CN2020/090168 2020-01-17 2020-05-14 一种利用古菌分子标记otu69快速检测城市搬迁地土壤全汞含量的方法 WO2021142988A1 (zh)

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