CN113122645B - Method for rapidly detecting effective calcium content in urban green land soil by using archaea molecular marker OTU384 - Google Patents
Method for rapidly detecting effective calcium content in urban green land soil by using archaea molecular marker OTU384 Download PDFInfo
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Abstract
The invention discloses a method for rapidly detecting the content of available calcium in urban green land soil by using an archaea molecular marker OTU 384. The invention provides a DNA molecule (probe) which is shown as a sequence 1 in a sequence table. The invention also protects the application of the probe in detecting or assisting in detecting the content of the available calcium in the soil. The invention also protects the application of the probe in comparing the effective calcium content of the soil in different plots. The method for detecting the content of the available calcium in the soil or comparing the content of the available calcium in the soil of different plots has the following advantages: the sample demand is little, need not the pretreatment, and the required time is short, and the human cost is low, can realize the quick automated inspection to big sample in batches. The method has important application and popularization values for soil sample evaluation.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for rapidly detecting the content of available calcium in urban green land soil by using an archaea molecular marker OTU 384.
Background
Urban landscaping construction is an important component of ecological civilization construction and beautiful Chinese construction, and plays an important role in optimizing urban environment, improving the quality of life of people and promoting urban sustainable development. The quality of the urban green land soil, which is taken as a material foundation of the urban ecological environment, directly influences the healthy growth of plants, the ecological benefit and the landscape function, and determines the quality of the urban green land to a certain extent. Therefore, scientific and objective monitoring and evaluation of the urban green land soil quality are important references and evidences for urban green land management.
At present, the traditional physical and chemical detection method is mainly used for detecting the urban green land soil quality index, the sample requirement is large, the sample pretreatment process is complex and long in period, particularly, the cost of required human resources is high, and the rapid detection of a large number of samples is difficult to realize. Soil microorganisms have the characteristics of comprehensiveness, sensitivity and functionality to changes of soil environment, and the exploration of the technology for realizing the rapid and automatic detection of the quality indexes of the green land soil by detecting the abundance of specific microorganism groups has important practical significance.
Calcium is a necessary nutrient element for plant growth, performs various physiological functions in plants, can be used as a signal substance to regulate cells, and can provide guarantee for normal operation of various metabolic activities in plants. There are 4 existing forms of calcium in soil, namely mineral calcium, organic calcium, exchange calcium and water-soluble calcium. Wherein the exchange state calcium and the water soluble state calcium are collectively called effective state calcium, and are available for plants.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting the effective calcium content in urban green land soil by using an archaea molecular marker OTU 384.
The invention provides a DNA molecule (probe) which is shown as a sequence 1 in a sequence table.
The invention specifically provides a Taqman probe, and a nucleotide sequence of the Taqman probe is shown as a sequence 1 in a sequence table. The 5 'end of the Taqman probe is provided with a fluorescent group, and the 3' end of the Taqman probe is provided with a fluorescent quenching group. The fluorophore may specifically be FAM. The fluorescence quenching group can be TAMRA.
The invention also protects the application of the DNA molecule or the Taqman probe in detecting or assisting in detecting the content of the effective calcium in the soil.
The invention also protects the application of the DNA molecule or the Taqman probe in comparison of the effective calcium content of soil in different plots.
The invention also protects a primer probe group, which consists of a specific primer pair and a specific probe; the specific primer pair consists of a primer 524F-10-ext shown in a sequence 4 of a sequence table and a primer Arch958R-mod shown in a sequence 5 of the sequence table; the nucleotide sequence of the specific probe is shown as a sequence 1 in a sequence table. The specific probe is a Taqman probe. The 5 'end of the Taqman probe is provided with a fluorescent group, and the 3' end of the Taqman probe is provided with a fluorescence quenching group. The fluorophore may specifically be FAM. The fluorescence quenching group can be TAMRA.
The invention also protects the application of the primer probe group in detecting or assisting in detecting the effective calcium content of soil.
The invention also protects the application of the primer probe set in comparing the effective calcium content of the soil of different plots.
The invention also provides a kit comprising the primer probe group.
The function of the kit is as follows (a) or (b):
(a) Detecting or assisting to detect the content of available calcium in the soil;
(b) Comparing the effective calcium content of the soil of different plots
The kit also comprises a carrier for recording the method A or the method B.
The invention also provides a method (method A) for detecting the content of available calcium in soil, which comprises the following steps:
taking the total DNA of the soil sample as a template, and carrying out real-time fluorescence quantitative PCR; the amplification primer pair of the real-time fluorescent quantitative PCR consists of a primer 524F-10-ext shown in a sequence 4 of a sequence table and a primer Arch958R-mod shown in a sequence 5 of the sequence table; the nucleotide sequence of the probe of the real-time fluorescence quantitative PCR is shown as a sequence 3 in a sequence table;
obtaining a Ct value by real-time fluorescence quantitative PCR; and calculating the copy number according to the Ct value, and calculating the effective calcium content in the soil sample according to the copy number content in the soil sample.
The probe of the real-time fluorescent quantitative PCR is a Taqman probe. The 5 'end of the Taqman probe is provided with a fluorescent group, and the 3' end of the Taqman probe is provided with a fluorescent quenching group. The fluorophore may specifically be FAM. The fluorescence quenching group can be TAMRA.
The copy number is the copy number of the target fragment of the probe.
The method for calculating the effective calcium content in the soil sample through the copy number content in the soil sample comprises the following steps: substituting the copy number content of the soil sample into a linear equation to obtain the effective calcium content of the soil sample.
The linear equation is: y = -15.345x+412.72; r 2 =0.9791; y represents available calcium contentAmount in mg/kg; x represents the copy number content in x 10 7 Number of copies/g.
The method for calculating the copy number according to the Ct value comprises the following steps: and substituting the Ct value into a standard curve equation to obtain the copy number.
The preparation method of the standard curve equation comprises the following steps: connecting a DNA molecule shown in a sequence 2 of a sequence table with a pMD18-T vector to obtain an OTU384 standard plasmid; the OTU384 standard quality grains were used to prepare a standard curve equation with the logarithm of the copy number as the independent variable and the Ct value as the dependent variable. The logarithm of the copy number is the base 10 logarithm of the copy number.
The preparation method of the total DNA of the soil sample comprises the following steps: using MoBio
DNA extraction kit (MoBio Laboratories, carlsbad, inc., CA, USA) extracts total DNA from soil samples.
Real-time fluorescent quantitative PCR
96 real-time fluorescent quantitative PCR instrument.
Reaction system for real-time fluorescent quantitative PCR (20 μ L): 10 μ L Premix Ex Taq (Takara, dalian, china), 0.4 μ L primer 524F-10-ext, 0.4 μ L primer Arch958R-mod, 0.2 μ L probe, 2 μ L template solution and 7 μ L sterile water. In the reaction system, the concentration of the primer 524F-10-ext was 0.2. Mu.M, the concentration of the primer Arch958R-mod was 0.2. Mu.M, and the concentration of the probe was 0.1. Mu.M. The amount of the template DNA in the reaction system was 7ng.
Reaction procedure for real-time fluorescent quantitative PCR: pre-denaturation at 95 ℃ for 120s; denaturation at 95 ℃ for 10s, annealing extension at 60 ℃ for 45s, and 45 cycles.
The invention also provides a method for comparing the effective calcium content of the soil in different plots (method B), which comprises the following steps:
respectively detecting soil samples of more than two plots according to the method A;
and comparing the effective calcium content of the soil of each land parcel according to the detection result.
Any one of the above soils is green soil.
Any of the above plots is a green plot.
Any one of the above soils is urban green land soil.
Any one of the land parcels is an urban green land parcel.
Any one of the above soils is green land soil of Yangtze river delta.
Any one of the land parcels is a green land parcel of Yangtze river delta.
Any one of the above soils is urban green land soil of Yangtze river delta.
Any one of the plots is an urban green plot of Yangtze river delta.
Any one of the above soils is park green land soil.
Any one of the land parcels is a park green land parcel.
Any one of the above soils is green soil of an urban park.
Any one of the land parcels is a green land parcel of a city park.
Any one of the above soils is park green land soil of Yangtze river delta.
Any one of the plots is a park green plot of Yangtze river delta.
Any one of the above soils is urban park green land soil of Yangtze river delta.
The land parcel is a green land parcel of an urban park of Yangtze river Delta.
The Yangtze river Delta is Shanghai city, jiangsu province and Zhejiang province.
Any of the above soil samples is taken from 0-20cm surface soil.
The method for detecting the content of the available calcium in the soil or comparing the content of the available calcium in the soil of different plots has the following advantages: the sample demand is little, need not the pretreatment, and the required time is short, and the human cost is low, can realize the quick automated inspection to big sample in batches. The method has important application and popularization values for soil sample evaluation.
Drawings
FIG. 1 is a linear relationship between copy number content of target OTU and soil available calcium content.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The soil quality index was determined according to relevant national, industrial and local standards, as detailed in table 1.
TABLE 1 soil quality index determination method
| Detecting the index | Detection method |
| pH | Determination of pH value of LY/T1239-1999 forest soil |
| Electrical conductivity of | LY/T1251-1999 forest soil water-soluble salinity analysis (conductance method) |
| Organic matter | Determination of soil organic matter of NY/T1121.6-2006 |
| Total nitrogen | LY/T1228-2015 forest soil nitrogen determination (Kjeldahl method) |
| Available nitrogen | LY/T1228-2015 forest soil nitrogen determination (alkaline hydrolysis-diffusion method) |
| All-phosphorus | LY/T1232-2015 forest soil phosphorus determination (alkali fusion molybdenum antimony colorimetric method) |
| Total potassium | Determination of LY/T1234-2015 forest soil Potassium (alkali fusion method) |
| Available phosphorus | DB 31/T661-2012 appendix F AB-DTPA leaching/inductively coupled plasma mass spectrometer |
| Effective potassium | DB 31/T661-2012 annex F AB-DTPA leaching/inductively coupled plasma emission spectrometer |
| Available sulfur | DB 31/T661-2012 appendix F AB-DTPA leaching/inductively coupled plasma mass spectrometer |
| Available chlorine | Reference in DB 31/T661-2012 appendix E (water saturated extraction) |
| Exchangeable sodium salt | Reference in DB 31/T661-2012 appendix E (water saturated extraction) |
| All arsenic | Atomic fluorescence method for measuring total mercury, total arsenic and total lead in GB/T22105.2-2008 soil quality |
| All copper | Full-digestion inductively coupled plasma mass spectrometerMethod of making |
| All zinc | Full digestion inductively coupled plasma mass spectrometer method |
| All-lead | Full digestion inductively coupled plasma mass spectrometer method |
| All-chromium | Full digestion inductive coupling plasma mass spectrometer method |
| All nickel | Same-above full-digestion inductively coupled plasma mass spectrometer method |
| Effective calcium | Refer to DB 31/T661-2012 annex F AB-DTPA leaching/inductively coupled plasma emission spectrometer |
| Effective manganese | DB 31/T661-2012 appendix F AB-DTPA leaching/inductively coupled plasma emission spectrometer |
| Effective zinc | DB 31/T661-2012 appendix F AB-DTPA leaching/inductively coupled plasma emission spectrometer |
| All mercury | USEPA 7473-2007 thermal decomposition homogeneous atomic absorption photometry |
Note: the only difference in effective calcium detection compared to the referenced method is that the detection target is effective calcium.
Example 1 OUT found to correlate with soil available calcium content
1. Collection of soil samples
In 11 months in 2017, study plots were set in a representative park green space of 16 administrative districts in Shanghai City.
The sampling method comprises the following steps: the collection of soil samples follows the sampling principle of multipoint mixing, 8 sampling points are selected for each sample land, 0-20cm of surface soil is collected by utilizing a soil drill with the diameter of 2.5cm, and then the surface soil is mixed into 1 soil sample.
A total of 76 soil samples were collected.
And (3) uniformly mixing the soil samples, sieving the mixture by using a 2mm sieve, and removing impurities such as plant roots, broken stones and the like. Each soil sample was then divided into two portions. Naturally air-drying one sample, and then sampling for determining the chemical property of the soil in the step two; another sample was stored at-80 ℃ and then sampled for total DNA extraction from soil in step three.
2. Analytical determination of soil quality indicators
And (5) analyzing and determining soil quality indexes of the naturally air-dried soil sample in the step one. The results of the soil quality measurements are shown in Table 2.
TABLE 2 soil quality index test results
| Detecting the index | Minimum value of | Maximum value | Mean value of |
| pH | 5.32 | 8.79 | 7.92 |
| Conductivity (μ S/cm) | 60.70 | 656.52 | 142.57 |
| Organic matter (g/kg) | 7.10 | 46.62 | 27.59 |
| Total nitrogen (g/kg) | 0.47 | 2.34 | 1.13 |
| Available nitrogen (mg/kg) | 25.85 | 152.64 | 84.68 |
| Total phosphorus (g/kg) | 0.42 | 1.00 | 0.68 |
| Total potassium (g/kg) | 15.80 | 25.43 | 19.08 |
| Available phosphorus (mg/kg) | 0.85 | 48.20 | 8.66 |
| Available potassium (m)g/kg) | 28.20 | 397.89 | 188.90 |
| Available sulfur (mg/kg) | 13.06 | 96.65 | 52.36 |
| Available chlorine (mg/L) | 4.16 | 1900.00 | 41.46 |
| Exchangeable sodium (mg/L) | 3.30 | 993.00 | 21.60 |
| Arsenic (mg/kg) | 4.81 | 13.50 | 8.73 |
| Copper (mg/kg) | 16.38 | 99.84 | 36.05 |
| Total zinc (mg/kg) | 87.38 | 223.87 | 125.52 |
| Whole lead (mg/kg) | 18.24 | 52.18 | 29.01 |
| Total chromium (mg/kg) | 55.40 | 101.00 | 72.90 |
| All nickel (mg/kg) | 27.40 | 44.59 | 36.62 |
| Effective calcium (mg/kg) | 198.36 | 366.95 | 282.89 |
| Available manganese (mg/kg) | 8.55 | 29.46 | 16.76 |
| Effective zinc (mg/kg) | 1.81 | 29.58 | 9.05 |
| Total mercury (mg/kg) | 0.05 | 0.57 | 0.21 |
3. Soil archaea population diversity analysis
1. And (4) extracting total DNA from the soil sample preserved at the temperature of minus 80 ℃ in the step one.
Extracting total DNA of soil sample by MoBio
DNA extraction kit (MoBio Laboratories, carlsbad, inc., CA, USA). And (3) repeatedly extracting each soil sample twice, and mixing the total DNA extracted twice to obtain a DNA sample. 76 soil samples were obtained, and 76 corresponding DNA samples were obtained. All DNA samples were stored at-80 ℃.
Using a Nanodrop 2000 ultramicro spectrophotometer and 0.8% agarose gel electrophoresis (5V cm) -1 45 min) detecting the DNA quality. The OD260/OD280 of 76 DNA samples are all in the range of 1.8-2.0, the maximum value of the OD260/OD280 is 1.98, and the minimum value of the OD260/OD280 is 1.81.
2. Archaea 16S rRNA gene amplification and high-throughput sequencing
A PCR amplification was carried out using a primer pair consisting of the primer 524F-10-ext and the primer Arch958R-mod, using the DNA sample as a template. After completing the PCR amplification, the PCR product was subjected to 2% agarose gel electrophoresis, and then the target band was gel-cut and purified using GeneJET gel recovery kit (Thermo Scientific), and then a sequencing library was constructed and sequenced using Illumina MiSeq sequencing platform (Illumina, san Diego, calif., USA).
The primers 524F-10-ext and the primer Arch958R-mod are archaea universal primers, and the target sequence is positioned in the V4-V5 variable region of the archaea 16S rRNA gene.
524F-10-ext (SEQ ID NO:4 of the sequence Listing): 5 '-tgycagcccgccggtaa-3';
arch958R-mod (SEQ ID NO:5 of the sequence Listing): 5 '-YCCGGCGTTGAVCCAATT-3';
y represents C or T; v represents G, A or C.
The reaction system for PCR amplification was 30. Mu.L. The effective component is 15 μ L
High-Fidelity PCR Master Mix (New England Biolabs), primers and template DNA. The concentration of each of the primers 524F-10-ext and the primer Arch958R-mod in the reaction system was 0.2. Mu.M. Inverse directionThe content of the template DNA in the reaction system was 10ng.
Reaction procedure for PCR amplification: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 33 cycles; extension at 72 ℃ for 5min.
3. High throughput data analysis and results
The bioinformatics analysis of high throughput sequencing results comprises the following steps: 1) Extracting the same sample sequence from the original data according to the sample specific label to form an independent file, and removing the label and the primer sequence; 2) Performing sequence splicing by using FLASH (V1.2.7) software; 3) Performing quality filtration on the original sequence after the sequencing is completed by using Qiime (V1.7.0) software; 4) Detecting chimera by using UCHIME software and deleting the chimera; 5) Partitioning the operational classification units (OTUs) at a similarity level of 95% using Uparse (v7.0.1001) software; 6) Because the reliability of the single copy sequence is questioned, the single copy sequence is removed in subsequent analyses; 7) In order to remove the influence of different sequencing depths among samples, the OTU tables of the samples are normalized to the same sequencing depth; 8) The OTUs were aligned based on the RDP database and their classification status was determined.
27765 archaea 16S rRNA gene sequences were selected for subsequent analysis for each sample. The archaea 16S rRNA gene sequences were classified under 95% sequence similarity, and a total of 580 OTUs were obtained.
4. Correlation analysis of soil archaea group and soil quality index
And (4) performing Pearson correlation analysis on the 580 OTUs obtained in the third step and each soil chemical index data obtained in the second step.
The results show that: among 580 OTUs, the correlation between the abundance of the archaea OTU384 in the soil and the content of the available calcium in the soil is strongest, and the correlation between the abundance of the archaea OTU447 in the soil and the content of the available calcium in the soil is ranked second; the abundance of the archaea OTU384 in the soil is obviously and negatively correlated with the content of the effective calcium in the soil, and the correlation coefficient r is-0.515; the correlation coefficient of the archaea OTU447 abundance in the soil and the effective calcium content in the soil is 0.335. The content of available calcium in the soil can be reflected by detecting the abundance of archaea OTU384 or archaea OTU447 in the soil.
Example 2 establishment of a Linear relationship between OTU and soil chemistry
1. Archaea Marker gene probe design
Based on the sequencing results, probes were designed for detection of OTU 384.
Probe384-1 Probe (sequence 1 of sequence table): 5 'TGAACAGGCTTAGTGCCTATT-doped 3'.
Probe384-2 Probe: 5 'AGTGCCTATTCAGTGCCGCA-3'.
The Probe384-1 Probe is a Taqman Probe, the 5 'end of the Probe is provided with a fluorescent group FAM, and the 3' end of the Probe is provided with a fluorescence quenching group TAMRA. The Probe384-2 Probe is a Taqman Probe, the 5 'end of the Probe is provided with a fluorescent group FAM, and the 3' end of the Probe is provided with a fluorescent quenching group TAMRA.
Based on the sequencing results, probes were designed for detection of OTU 447.
Probe 447-1: 5 'AGGTGTCGACTCCTTCCGTT-3'.
Probe447-2 Probe: 5 'AGCTAACGCATTAAGCG-3'.
The Probe447-1 is a Taqman Probe, the 5 'end of which is provided with a fluorescent group FAM and the 3' end of which is provided with a fluorescent quenching group TAMRA. The Probe447-2 Probe is a Taqman Probe, the 5 'end of the Probe is provided with a fluorescent group FAM, and the 3' end of the Probe is provided with a fluorescent quenching group TAMRA.
2. Determination of archaea Marker gene copy number in soil
According to the measured value, soil samples with gradient distribution of effective calcium content are randomly selected from the soil samples in the first step of example 1.
1. Taking a soil sample, and extracting total DNA.
The total DNA was extracted in the same manner as in step three 1 of example 1.
2. Taking the template solution (i.e., the total DNA solution obtained in step 1), and using
The 96 real-time fluorescent quantitative PCR instrument performs fluorescent quantitative PCR (probe method).
A primer pair consisting of the primer 524F-10-ext and the primer Arch958R-mod was used. Probe384-1 Probe or Probe384-2 Probe or Probe447-1 Probe or Probe447-2 Probe was used.
Reaction system (20 μ L): 10 μ L of Premix Ex Taq (Takara, dalian, china), 0.4 μ L of primer 524F-10-ext, 0.4 μ L of primer Arch958R-mod, 0.2 μ L of probe, 2 μ L of template solution and 7 μ L of sterile water. In the reaction system, the concentration of the primer 524F-10-ext was 0.2. Mu.M, the concentration of the primer Arch958R-mod was 0.2. Mu.M, and the concentration of the probe was 0.1. Mu.M. The amount of the template DNA in the reaction system was 7ng.
Reaction procedures are as follows: pre-denaturation at 95 ℃ for 120s; denaturation at 95 ℃ for 10s, annealing extension at 60 ℃ for 45s, and 45 cycles.
The amplification specificity was determined by a lysis curve.
And substituting the Ct value into a standard curve equation to obtain the copy number of the target OTU, and calculating to obtain the copy number content (the unit is copy number/g, namely the copy number of the target OTU in the soil sample per g dry weight) of the target OTU in the soil sample.
The standard curve equation is prepared in step 3.
3. Preparation of fluorescent quantitative PCR Standard Curve
Connecting a DNA molecule (obtained by amplifying from total soil DNA) shown in a sequence 2 of the sequence table with a pMD18-T vector to obtain an OTU384 standard plasmid. Preparing standard quality particle solutions containing OTU384 standard plasmids with different concentrations by using TE buffer solution as a solvent (determining the DNA concentration in the standard quality particle solution by using a Nanodrop 2000 ultramicro spectrophotometer, and converting the DNA copy number to obtain the OTU384 copy number); each standard plasmid solution was used as a template solution, and the detection was performed according to the method of step 2 (using Probe384-1 Probe or Probe384-2 Probe), to obtain a standard curve equation with logarithm of the copy number of OTU384 (logarithm to base 10) as an independent variable and Ct value as a dependent variable.
Connecting a DNA molecule (obtained by amplifying total soil DNA) shown in a sequence 3 in a sequence table with a pMD18-T vector to obtain an OTU447 standard plasmid. Preparing standard quality particle solutions containing OTU447 standard plasmid with different concentrations by using TE buffer solution as a solvent (determining the DNA concentration in the standard quality particle solution by a Nanodrop 2000 ultramicro spectrophotometer, and converting the DNA copy number to obtain the OTU447 copy number); each standard plasmid solution was used as a template solution, and the detection was performed according to the method of step 2 (using Probe447-1 Probe or Probe447-2 Probe) to obtain a standard curve equation with the logarithm of the OTU447 copy number (the logarithm to the base 10) as an independent variable and the Ct value as a dependent variable.
Fluorescent quantitative PCR amplification was between 90% and 110% efficient.
3. Establishment of linear relation between archaea Marker gene copy number and soil chemical characteristics
The results of the measured effective calcium value (in mg/kg), ct value and the copy number content (in copy number/g) of the target OTU of the soil sample are shown in Table 3. The measured value of the effective calcium of the soil sample is the data obtained in step two 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 two of this example. The Probe384-1 Probe is used for carrying out fluorescence quantitative PCR, and the relative abundance of the target OTU in the soil (expressed as the copy number of the target OTU in the soil) and the effective calcium content in the soil have a good linear relationship.
TABLE 3
Note: soil sample 1, collected from park green land in the new region of Pudong; a soil sample 2 collected from a park green land in a gold mountain area; a soil sample 3 collected from a park greenbelt in a Minkou area; a soil sample 4 collected from a park green land of a Putuo district; soil sample 5, collected from park greenfield in the iris area; soil sample 6, collected from park greenfield in Pudong New district.
The Probe384-1 Probe is used for carrying out fluorescence quantitative PCR, and the linear relation between the copy number content of the target OTU in the soil and the effective calcium content of the soil is shown in figure 1. The linear equation is: y = -15.345x +, 412.72; r is 2 =0.9791; y represents the available calcium content (mg/kg), and x represents the copy number content of the target OUT (× 10) 7 Copy number/g).
Example 3 molecular detection of effective calcium content in unknown urban Green land soil samples
3 green lands are randomly selected in Shanghai plant garden, nanjing basalt lake park and Suzhou Wujiang park respectively.
The sampling method comprises the following steps: the collection of soil samples follows the sampling principle of multipoint mixing, 8 sampling points are selected for each sample land, 0-20cm of surface soil is collected by utilizing a soil drill with the diameter of 2.5cm, and then the surface soil is mixed into 1 soil sample.
And (3) uniformly mixing the soil samples, sieving the soil samples by using a 2mm sieve, and removing impurities such as plant roots, broken stones and the like. Each soil sample was then divided into two portions. One sample is naturally dried, and then is sampled to detect the content of effective calcium, so as to obtain the measured value (unit is mg/kg) of the content of effective calcium; another sample was stored at-80 ℃ and then sampled for soil total DNA extraction.
1. Extracting the total DNA of the soil sample. Using MoBio
DNA extraction kit (MoBio Laboratories, carlsbad, inc., CA, USA). And (3) repeatedly extracting each soil sample twice, and mixing the total DNA extracted twice to obtain a DNA sample.
2. Taking the template solution (i.e., the DNA sample obtained in step 1), and using it
The 96 real-time fluorescent quantitative PCR instrument performs fluorescent quantitative PCR (probe method).
Reaction system (20 μ L): mu.L of Premix Ex Taq (Takara, dalian, china), 0.4. Mu.L of primer 524F-10-ext, 0.4. Mu.L of primer Arch958R-mod, 0.2. Mu.L of Probe384-1 Probe, 2. Mu.L of template solution and 7. Mu.L of sterile water. In the reaction system, the concentration of the primer 524F-10-ext was 0.2. Mu.M, the concentration of the primer Arch958R-mod was 0.2. Mu.M, and the concentration of the Probe384-1 Probe was 0.1. Mu.M. The amount of the template DNA in the reaction system was 7ng.
Reaction procedures are as follows: pre-denaturation at 95 ℃ for 120s; denaturation at 95 ℃ for 10s, annealing extension at 60 ℃ for 45s, and 45 cycles.
And substituting the Ct value into a standard curve equation to obtain the copy number of the OTU384, and calculating to obtain the copy number content (the unit is the copy number/g, namely the copy number of the OTU384 in each g of dry weight of the soil sample) of the OTU384 in the soil sample.
The standard curve equation was generated as described in step two, 3, of example 2.
3. Substituting the copy number content of OTU384 in the soil sample into a linear equation to obtain a calculated value (unit is mg/kg) of the effective calcium content of the soil sample.
The linear equation is: y = -15.345x+412.72; r 2 =0.9791; y represents the available calcium content (mg/kg), and x represents the copy number content of OTU384 (x 10) 7 Copy number/g).
The results of the copy number content (in copy number/g) of OTU384 in the soil sample, the calculated value (in mg/kg) of the effective calcium content of the soil sample, and the measured value (in mg/kg) of the effective calcium content of the soil sample are shown in table 4.
TABLE 4
SEQUENCE LISTING
<110> research institute for garden science planning in Shanghai City
<120> method for rapidly detecting effective calcium content in urban green land soil by using archaea molecular marker OTU384
<130> GNCYX200029
<160> 5
<170> PatentIn version 3.5
<210> 1
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<213> Artificial sequence
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tgaacaggct tagtgcctat t 21
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tgtcagccgc cgcggtaata ccagcacccc gagtggtcgg gacgattatt gggcctaaag 60
catccgtagc cggttctaca agtcttccgt taaatccacc tgcttaacag atgggctgca 120
gaggatacta tagagctagg aggcgggaga ggcaagcggt acttagtggg taggggtaaa 180
atccgttgat ccactgaaga ccaccagtgg cgaaggcggc ttgccagaac gcgctcgacg 240
gtgagggatg aaagctgggg gagcaaaccg gattagatac ccgggtagtc ccagctgtaa 300
acgatgcaga ctcggtgatg aacaggctta gtgcctattc agtgccgcag ggaagccgtt 360
aagtctgccg cctggggagt acggtcgcaa gactgaaact taaaggaatt ggcgggggag 420
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tgtcagccgc cgcggtaata ccagcacccc gagtggtcgg gacgattatt gggcctaaag 60
catccgtagc cggtcctgca agtcctccgt taaatccacc cgctcaacgg ttgggccgcg 120
ggggatacta cagggctagg aggcgggaga ggcaagcggt actcgatggg taggggtaaa 180
atccactgat ccattgaaga ccaccagtgg cgaaggcggc ttgccagaac gcgctcgacg 240
gtgagggatg aaagctgggg gagcaaaccg gattagatac cctggtagtc cacgccgtaa 300
acgatgggcg ctaggtgtgg gaggtgtcga ctccttccgt gccgcagcta acgcattaag 360
cgccccgcct ggggagtacg gtcgcaagac tgaaacttaa aggaattggc gggggagcac 420
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Claims (6)
1. The primer probe group consists of a specific primer pair and a specific probe; the specific primer pair consists of SEQ ID NO:4 and primers 524F-10-ext shown in SEQ ID NO:5, and the primer Arch 958R-mod; the nucleotide sequence of the specific probe is shown as SEQ ID NO:1 is shown.
2. The primer probe set of claim 1 is used for detecting or assisting in detecting the content of available calcium in soil; the soil is green land soil of Yangtze river delta.
3. Use of the primer probe set of claim 1 for comparing the available calcium content of soil in different plots; the land parcel is a green land parcel of Yangtze river delta.
4. A kit comprising the primer probe set of claim 1.
5. A method for detecting the effective calcium content of soil comprises the following steps:
taking total DNA of a soil sample as a template, and carrying out real-time fluorescent quantitative PCR; the amplification primer pair of the real-time fluorescent quantitative PCR consists of SEQ ID NO:4 and primers 524F-10-ext shown in SEQ ID NO:5, in the sequence shown in the specification, and consists of the primer Arch 958R-mod; the nucleotide sequence of the probe for real-time fluorescent quantitative PCR is shown as SEQ ID NO:1 is shown in the specification;
ct value is obtained by real-time fluorescence quantitative PCR; calculating copy number according to the Ct value, and calculating the copy number content in the soil sample to obtain the effective calcium content in the soil sample;
the soil is green land soil of Yangtze river delta.
6. A method for comparing the effective calcium content of soil in different plots comprises the following steps:
testing soil samples of more than two plots respectively according to the method of claim 5;
comparing the effective calcium content of the soil of each land parcel according to the detection result;
the land parcel is a green land parcel of Yangtze river delta.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368214A (en) * | 2008-09-24 | 2009-02-18 | 上海大学 | Primer for archeobacteria heterogeneity analysis and aiming at 16S rRNA gene |
| WO2013004933A1 (en) * | 2011-07-05 | 2013-01-10 | Assistance Publique - Hopitaux De Marseille | Culturing and detection of a methanogenic archaeon |
| CN107190055A (en) * | 2017-04-28 | 2017-09-22 | 浙江大学 | Soil bacteria high flux absolute quantification method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101368214A (en) * | 2008-09-24 | 2009-02-18 | 上海大学 | Primer for archeobacteria heterogeneity analysis and aiming at 16S rRNA gene |
| WO2013004933A1 (en) * | 2011-07-05 | 2013-01-10 | Assistance Publique - Hopitaux De Marseille | Culturing and detection of a methanogenic archaeon |
| CN107190055A (en) * | 2017-04-28 | 2017-09-22 | 浙江大学 | Soil bacteria high flux absolute quantification method |
Non-Patent Citations (2)
| Title |
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| "Revegetation of urban green space rewilds soil microbiotas with implications for human health and urban design";Jacob G. Mills et al.;《Restoration Ecology》;20200930;第28卷(第S4期);第S322-S334页 * |
| "高钙废水颗粒污泥中古菌菌群结构变化的分析";张安龙等;《环境科学学报》;20191231;第39卷(第12期);第3956-3965页 * |
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