Patent Application
20220220537
The present 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 using archaea molecular marker OTU69.
With the rapid development of urbanization, cities, especially megacities, have entered a stage of urban development dominated by “urban redevelopment”. A large number of urban relocation sites have appeared, especially in cities in economically developed areas along the eastern coast. Taking Shanghai as an example, 80%-90% of the green land in the built-up area was developed on the demolition of urban villages and the relocation of old factories. Scientific and objective monitoring and evaluation for its soil quality is a prerequisite and an important reference basis for ecological restoration and landscaping in urban relocation sites.
At present, the detection of soil quality indicators in urban relocation sites mainly follows traditional physical and chemical detection methods, which require a lot of samples, a complicated pretreatment process, a long cycle, and a high cost on human resources. So it is difficult to achieve rapid detection of large quantities of samples. Soil microorganisms have comprehensive, sensitive, and functional characteristics to the changes in the soil environment. It is of great practical significance to explore the rapid and automatic detection technology for soil quality indicators in urban relocation sites by detecting the abundance of specific microbial groups.
Element mercury is one of the most harmful heavy metals to the human body and is one of the 129 priority control pollutants. The mercury in the soil mainly comes from 1) The soil parent material, the mercury in the soil parent material is the basic source in the soil. The content of mercury in the primary rock directly determines the content of mercury in the soil. 2) Atmospheric deposition, after the mercury in the atmosphere enters the soil, most of it is quickly absorbed or fixed by the clay minerals and organic matter in the soil and enriched in the surface of the soil. 3) Direct pollution, mainly including the stacking of industrial production waste and urban domestic garbage, unreasonable application of mercury-containing fertilizers and pesticides, irrigation, etc. 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 the soil in urban relocation sites by using archaea molecular marker OTU69.
The present invention also provides a method (Method A) for detecting total mercury content in the soil, which includes the following steps:
Using the total DNA of the soil sample as a template to perform real-time fluorescent quantitative PCR; The amplification primer pair of real-time fluorescence quantitative PCR is composed of primer 524F-10-ext shown as SEQ ID NO.4 of the sequence listing and primer Arch958R-mod shown as SEQ ID NO.5 of the sequence listing. The nucleotide sequence of the probe for real-time fluorescence quantitative PCR is shown as SEQ ID NO.1 of the sequence listing.
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 linear equation is y=−0.1811x+0.6508; y represents total mercury content, and the unit is mg/kg; x represents copy number content, and the unit is ×107 copies/g. R2=0.959 for the linear equation.
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: ligate the DNA molecule shown as SEQ ID NO.2 of the sequence listing to the pMD18-T vector to obtain OTU69 standard plasmid; and use the OTU69 standard plasmid to produce a standard curve equation with the logarithm of the copy number as the independent variable and Ct as the dependent variable. The logarithm of the copy number is the logarithm with base 10 of the copy number.
Preparation method of total DNA of soil samples: AdoptMoBioPowerSoil® DNA Isolation Kit (MoBio Laboratories, Carlsbad, Inc., CA, USA) to extract total DNA of soil samples.
The real-time fluorescent quantitative PCR was performed on aLightCycler® 96 real-time fluorescent quantitative PCR instrument.
The real-time fluorescent quantitative PCR reaction system (20 μL) is: 10 μL Premix Ex Taq (Takara, Dalian, China), 0.4 μL primer 524F-10-ext, 0.4 μL primer Arch958R-mod, 0.2 μL probe, 2 μL template solution and 7 μL sterile water. In the reaction system, the concentration of primer 524F-10-ext is 0.2 μM, wherein the concentration of primer Arch958R-mod is 0.2 μM, and the concentration of probe is 0.1 μM. In the reaction system, the content of template DNA is Ing.
The real-time fluorescent quantitative PCR reaction program: pre-denaturation at 95° C. for 120 s, denaturation at 95° C. for 10 s, annealing extension at 60° C. for 45 s, 45 cycles.
The present invention also provides a method for comparing the total mercury content of the soil in different plots (Method B), which includes the following steps:
testing the soil samples of more than two plots according to Method A;
comparing the total mercury content in the soil of each plot based on the test results.
The present invention provides a DNA molecule (probe), shown in SEQ ID NO.1 of the sequence listing.
The DNA molecule (probe) 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. The label includes but not limited to dyes; radiolabels, such as 32P; binding moieties, such as biotin; haptens, such as digoxin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or fluorescent dyes combined with moieties, of which the emission spectrum can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The labels can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, and so on. Labels can be a charged moiety (positive or negative), or optionally can be neutral. Labels may include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing labels is detectable. In some embodiments, directly detect the nucleic acid without the label (e.g., read the sequence directly).
The present invention provides a Taqman probe, its nucleotide sequence shown as SEQ ID NO.1 of the sequence listing. 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 present invention also claims the application of the DNA molecule or the Taqman probe in detecting or assisting in detecting the total mercury content of the soil.
The present invention also claims 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 claims the primer-probe set, which is composed of a specific primers pair and a specific probe; The specific primers pair is composed of the primer 524F-10-ext shown in SEQ ID NO.4 of the sequence listing and the primer Arch958R-mod shown in SEQ ID NO.5 of the sequence listing; The nucleotide sequence of the specific probe is shown in SEQ ID NO.1 of the sequence listing. The specific probe is a Taqman probe. The Taqman probe has a fluorescent group at its 5′end and a fluorescence quenching group at its 3′end. The fluorescent group may specifically be FAM. The fluorescence quenching group may specifically be TAMRA.
The present invention also protects the application of the primer-probe set in detecting or assisting in detecting the total mercury content of the soil.
The present 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 said primer-probe set.
The function of the kit is as the following (a) or (b):
(a) Detecting or assisting in detecting the total mercury content of the soil;
(b) Comparing the total mercury content of the soil in different plots.
The kit also includes a carrier recording the Method A or the Method B.
Any of the above-mentioned soil is green land soil.
Any of the above-mentioned plots is a green land plot.
Any of the above-mentioned soil is green land soil in China.
Any of the above-mentioned plots is a green land plot in China.
Any of the above-mentioned soil is urban green land soil.
Any of the above-mentioned plots is an urban green land plot.
Any of the above-mentioned soil is green land soil in Yangtze River Delta of China.
Any of the above-mentioned plots is a green land plot in Yangtze River Delta of China.
Any of the above-mentioned soil is urban green land soil in Yangtze River Delta of China.
Any of the above-mentioned plots is an urban green land plot in Yangtze River Delta of China.
Any of the above-mentioned soil is park green land soil.
Any of the above-mentioned plots is a park green land plot.
Any of the above-mentioned soil is urban park green land soil.
Any of the above-mentioned plots is a urban park green land plot.
Any of the above-mentioned soil is park green land soil in Yangtze River Delta of China.
Any of the above-mentioned plots is a park green land plot in Yangtze River Delta of China.
Any of the above-mentioned soil is urban park green land soil in Yangtze River Delta of China.
Any of the above-mentioned plots is a urban park green land plot in Yangtze River Delta of China.
Any of the above-mentioned soil is relocation site soil.
Any of the above-mentioned plots is a relocation site plot.
Any of the above-mentioned soil is relocation site soil in China.
Any of the above-mentioned plots is a relocation site plot in China.
Any of the above-mentioned soil is urban relocation site soil.
Any of the above-mentioned plots is an urban relocation site plot.
Any of the above-mentioned soil is relocation site soil in the Yangtze River Delta of China.
Any of the above-mentioned plots is a relocation site plot in the Yangtze River Delta of China.
Any of the above-mentioned soil is urban relocation site soil in the Yangtze River Delta of China.
Any of the above-mentioned plots is an urban relocation site plot in the Yangtze River Delta of China.
The Yangtze River Delta refers to China's Shanghai, Jiangsu Province, and Zhejiang Province.
Any of the above-mentioned soil samples are taken from 0-20 cm surface 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. The experimental 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.
The soil quality indicators are determined according to relevant national standards, industry standards and local standards, see Table 1 for details.
Note: Compared with the reference method, the only difference in available calcium detection is that the detection target is available calcium.
1. Collection of Soil Samples
In November 2017, research sample plots were set up in representative parks and green lands in 16 administrative regions of Shanghai, China.
Sampling method: The collection of soil samples followed the principle of multi-point mixing. Choose 8 sampling points in each plot, use a 2.5 cm diameter soil drill to collect 0-20 cm surface soil, and then mix them into one soil sample.
A total of 76 soil samples were collected.
The soil samples were mixed evenly and passed through a 2 mm sieve to remove plant roots, gravel, and other debris. Then each soil sample was divided into two partitions. One partition was air-dried naturally, and then as the sample for the determination of soil chemical properties in step 2; the other partition was stored at −80° C., and then as the sample used for the extraction of total soil DNA in step 3.
2. Analysis and Determination of Soil Quality Indicators
In step 1, the natural air-dried soil samples were analyzed and determined for soil quality indicators. The measurement results of soil quality indicators were shown in Table 2.
3. Diversity Analysis of Soil Archaea Population
(1). Extraction of Total DNA from Soil Samples Stored at −80° C. in Step 1.
Total DNA of soil samples was extracted with MoBioPowerSoil® DNA Isolation Kit (MoBio Laboratories, Carlsbad, Inc., CA, USA). Each soil sample was extracted twice, and the total DNA extracted twice was mixed to obtain a DNA sample. For 76 soil samples, corresponding 76 DNA samples were obtained. All DNA samples were stored at −80° C.
The DNA quality was examined by Nanodrop 2000 ultra-micro spectrophotometer and 0.8% agarose gel electrophoresis (5V cm′, 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.
(2). Archaea 16S rRNA Gene Amplification and High-Throughput Sequencing
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. After PCR amplification was completed, the PCR products were subjected to 2% agarose gel electrophoresis, and then the target bands were cut and purified by the GeneJET Gel Recovery Kit (Thermo Scientific), and then the sequencing library was constructed, and the Illumina MiSeq sequencing platform (Illumina, San Diego, Calif., USA) was used for sequencing.
Primer 524F-10-ext and primer Arch958R-mod are universal primers for archaea, and the target sequence is located in the V4-V5 variable region of the archaea 16S rRNA gene.
Y stands for C or T; V stands for G, A or C.
The reaction system for PCR amplification was 30 μL. The active ingredients were 15 μL, of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), primers and template DNA. In the reaction system, the concentration of primer 524F-10-ext and primer Arch958R-mod were both 0.2 μM. In the reaction system, the content of template DNA was 10 ng.
The PCR amplification reaction program: 95° C. pre-denaturation 3 min; 95° C. denaturation 30 s, 55° C. annealing 30 s, 72° C. extension 45 s, 33 cycles; 72° C. extension 5 min
(3). High-Throughput Data Analysis and Results
The specific steps for bioinformatics analysis of high-throughput sequencing results were as follows: 1) Extracted the same sample sequence from the original data according to the sample-specific label to form a separate file, and removed the label and primer sequences; 2) FLASH (V1.2.7) software was used for sequence splicing; 3) Qiime (V1.7.0) software was used to perform quality filtering on the original sequence after sequencing; 4) UCHIME software was used to detect chimeras and deleted them; 5) Uparse (v7.0.1001) software was used to divide Operational Taxonomic Units (OTUs) at a similarity level of 95%; 6) Since the reliability of the single-copy sequence was questioned, the single-copy sequence was removed in the subsequent analysis; 7) In order to remove the influence of different sequencing depths between samples, the sample OTU table was homogenized to the same sequencing depth; 8) Aligned the sequence of OTUs based on the RDP database and determined the taxonomic status thereof.
Total 27765 archaea 16S rRNA gene sequences were selected from each sample for subsequent analysis. The archaea 16S rRNA gene sequences were classified at 95% sequence similarity, and a total of 580 OTUs were obtained.
4. Correlation Analysis Between Soil Archaea Groups and Soil Quality Indicators
Performed Pearson correlation analysis on the 580 OTUs obtained in step 3 and the soil chemical indicators data obtained in step 2.
The results show that among 580 OTUs, the abundance of archaeal OTU69 in the soil has the strongest correlation with the total mercury content in the soil; the correlation between the abundance of archaea OTU384 in the soil and the total mercury content of the soil comes second; meanwhile the abundance of archaea OTU69 in the soil was significantly negatively correlated with the total mercury content of the soil, 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 archaea OTU384 in the soil can be used to reflect the total mercury content of the soil.
1. Archaea Marker Gene Probe Design
According to the sequencing results, a probe for detecting OTU69 was designed.
The Probe69-1 was a Taqman probe with a fluorescent group FAM at its 5′end and a fluorescence quenching group TAMRA at its 3′end. The Probe69-2 was a Taqman probe with a fluorescent group FAM at its 5′end and a fluorescence quenching group TAMRA at its 3′end.
According to the sequencing results, a probe for detecting OTU384 was designed.
The Probe384-1 was a Taqman probe with a fluorescent group FAM at its 5′end and a fluorescence quenching group TAMRA at its 3′end. The Probe384-2 was a Taqman probe with a fluorescent group FAM at its 5′end and a fluorescence quenching group TAMRA at its 3′end.
2. Determination of the Copy Number of Archaea Marker Gene in Soil
According to the measured value, soil samples with a gradient distribution of total mercury content were randomly selected from the soil samples in step 1 of Example 1.
(1) Took soil samples for extraction of the total DNA.
The extraction method of the total DNA was the same as that of step 3 of Example 1.
(2) Took the template solution (that was, the total DNA solution obtained in step 1), and performed fluorescent quantitative PCR (probe method) on the LightCycler® 96 real-time fluorescent quantitative PCR instrument.
A primer pair consisting of the primer 524F-10-ext and the primer Arch958R-mod was used. Probe69-1 probe or Probe69-2 probe or Probe384-1 probe or Probe384-2 probe was used.
Reaction system (20 μL): 10 μL, Premix Ex Taq (Takara, Dalian, China), 0.4 μL, primer 524F-10-ext, 0.4 μL, primer Arch958R-mod, 0.2 μL, probe, 2 μL template solution and 7 μL sterile water. In the reaction system, the concentration of primer 524F-10-ext was 0.2 μM, the concentration of primer Arch958R-mod was 0.2 μM, and the concentration of probe was 0.1 μM. In the reaction system, the content of template DNA was Ing.
Reaction program: 95° C. pre-denaturation for 120 s; 95° C. denaturation for 10 s, 60° C. annealing extension for 45 s, 45 cycles.
The specificity of amplification was determined by the melting curve.
The Ct value was substituted into the standard curve equation to obtain the copy number of the target OTU, and then the copy number content (unit was copy number/g, that was, the copy number of the target OTUin each gram of the dry soil sample) of the target OTU in the soil sample was calculated.
The preparation method of the standard curve equation was shown in step 3.
(3) Constructed a standard curve for real-time PCR
Ligated the DNA molecule (amplified from total soil DNA) shown as SEQ ID NO.2 of the sequence listing to the pMD18-T vector to obtain OTU69 standard plasmid. Using TE buffer as the solvent, prepared the standard plasmid solutions containing different concentrations of OTU69 standard plasmid (the DNA concentration in the standard plasmid solution was measured by Nanodrop 2000 ultra-micro spectrophotometer, and was converted into DNA copy number, which was the OTU69 copy number). Each standard plasmid solution was used as a template solution, and detection was performed according to the method of step 2 (using the Probe69-1 probe or the Probe69-2 probe) to obtain the standard curve equation with the logarithm of OTU69 copy number (logarithm with base 10) as the independent variable and Ct as the dependent variable.
CTCAACGGTTGGGCTGCGGAGGATACTACGTGGCTAGGAGGCGGGAGAGGC
Ligated the DNA molecule (amplified from total soil DNA) shown as SEQ ID NO.3 of the sequence listing to the pMD18-T vector to obtain OTU384 standard plasmid. Using TE buffer as the solvent, prepared standard plasmid solutions containing different concentrations of OTU384 standard plasmid (the DNA concentration in the standard plasmid solution was measured by Nanodrop 2000 ultra-micro spectrophotometer, and was converted into DNA copy number, which was the OTU384 copy number). Each standard plasmid solution was used as a template solution, and detection was performed according to the method of step 2 (using the Probe384-1 probe or the Probe384-2 probe) to obtain the standard curve equation with the logarithm of OTU384 copy number (logarithm with base 10) as the independent variable and Ct as the dependent variable.
The efficiency of fluorescence quantitative PCR amplification was between 90% and 110%.
3. Establishment of a Linear Relationship Between Copy Number of Archaea Marker Gene and Chemical Characteristics of the Soil
The results of the measured total mercury values (unit: mg/kg), Ct values, and target OTU copy number content (unit: copy number/g) of the soil samples were shown in Table 3. The measured values of total mercury of the soil samples were the data obtained in step 2 of Example 1. The Ct values and the copy number content of the target OTU of the soil samples were the data obtained in step 2 of the present embodiment. Probe69-1 probe was used to perform fluorescence quantitative PCR, the relative abundance of target OTU of the soil (reflected as the copy number of target OTU in the soil) had a good linear relationship with the total mercury content of the soil.
Note: soil sample 1, collected from a green land of the park in Songjiang District; soil sample 2, collected from a green land of the park in Jinshan District; soil sample 3, collected from a green land of the park in Huangpu District; soil sample 4, collected from a green land of the park in Jing'an District; soil sample 5, collected from a green land of the park in Putuo District; soil sample 6, collected from a green land of the park in Minhang District.
Probe69-1 probe was used to perform fluorescence quantitative PCR, the linear relationship between the copy number content of the target OTU in the soil and the total mercury content of the soil was shown in
Randomly selected three green lands of urban parks in Shanghai, Nanjing and Suzhou in China.
Sampling method: The collection of soil samples followed the principle of multi-point mixing: selected 8 sampling points in each plot, collected 0-20 cm surface soil using a 2.5 cm diameter soil drill, and then mix the collected soil into one soil sample.
The soil samples were mixed evenly and passed through a 2 mm sieve to remove plant roots, gravel, and other debris. Then each soil sample was divided into two parts. One part was air-dried naturally, and then as a sample to be detected to get the actual measured value of the total mercury content (unit: mg/kg). The other part was stored at −80° C. and then as a sample for extraction of total soil DNA.
1. Extraction of total DNA of soil samples. Adopted MoBioPowerSoil® DNA Isolation Kit (MoBio Laboratories, Carlsbad, Inc., CA, USA). Extracted each soil sample two times and mix all of the extracted total DNA to get a DNA sample.
2. Took the template solution (i.e. the DNA sample obtained in step 1), and performed fluorescent quantitative PCR (probe method) on the LightCycler® 96 real-time fluorescent quantitative PCR instrument.
Reaction system (20 μL): 10 μL, Premix Ex Taq (Takara, Dalian, China), 0.4 μL, primer 524F-10-ext, 0.4 μL, primer Arch958R-mod, 0.2 μL, Probe69-1 probe, 2 μL, template solution and 7 μL, sterile water. In the reaction system, the concentration of primer 524F-10-ext was 0.2 μM, the concentration of primer Arch958R-mod was 0.2 μM, and the concentration of Probe69-1 probe was 0.1 μM. In the reaction system, the content of template DNA was Ing.
Reaction program: 95° C. pre-denaturation for 120 s; 95° C. denaturation for 10 s, 60° C. annealing extension for 45 s, 45 cycles.
The Ct value was substituted into the standard curve equation to obtain the copy number of OTU69, and then the copy number content of OTU69 of the soil sample was calculated (the unit was copy number/g, that was, the number of copies of OTU69 per gram of dry weight of soil sample).
The construction method of the standard curve equation was shown in step (3) of part 2 of Example 2.
3. The copy number content of OTU69 of the soil sample was substituted into the linear equation to obtain the calculated value of the total mercury content of the soil sample (unit: mg/kg).
The linear equation is: y=−0.1811x+0.6508; R2=0.959; wherein y represents the total mercury content (mg/kg), x represents the copy number content of OTU69 (×107 copies/g).
The results of the copy number content of OTU69 in the soil sample (unit: copy number/g), the calculated value of the total mercury content of the soil sample (unit: mg/kg), and the measured value of the total mercury content of the soil sample (unit: mg/kg) are shown in Table 4.
In Shanghai, Nanjing, and Suzhou in China, three city relocation sites were randomly selected.
The method was the same as in Example 3.
The results of the copy number content of OTU69 in the soil sample (unit: copy number/g), the calculated value of the total mercury content of the soil sample (unit: mg/kg), and the measured value of the total mercury content of the soil sample (unit: mg/kg) are shown in Table 5.
The invention discloses a method for detecting the total mercury content of the soil or comparing the total mercury content of the soil between different plots, which has the following advantages: detecting the total mercury content of the soil or comparing the total mercury content of the soil between different plots, and small sample demand, no need for pre-treatment, short required time, low labor cost, realizing the rapid automatic detection of large quantities of samples. The present invention deserves to apply and promote in the evaluation of soil samples.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010051314.X | Jan 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/090168 | 5/14/2020 | WO | 00 |
