CN107058580A - A kind of microorganisms in water pollution source Methodology of Quantitative Analysis - Google Patents
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Abstract
The invention discloses a kind of microorganisms in water pollution source Methodology of Quantitative Analysis, choose have high specificity, sensitivity high for target area and can quantitative analysis specific biomarker primer, pass through qPCR amplification techniques and build calibration curve equation.Actual waters sample is gathered again, extracts the genomic DNA in water sample；QPCR amplifications are carried out using region-specific primers, calibration curve equation is as a result substituted into and is calculated, the concentration value of each microorganism in target water is drawn, according to the concentration value of each microorganism come the pollution sources of resolution areas target water.The objective microbe of low concentration can recognize that by the method for the present invention, it is adaptable to which the environment water of different quality, for the monitoring of environment water, tracing to the source and administering provides reliable foundation.
Description
Technical field
This patent disclosure relates generally to environmental monitoring technology, and in particular to a kind of microorganisms in water pollution source quantitative resolution side
Method.
Background technology
Unprocessed (or incompletely processed) people and Animal manure loss are the main originals for causing microorganisms in water to pollute
Cause, how quick and precisely distinguishing in water body the main source of microorganism pollution and timely and effectively carrying out specific aim Sources controlling is
The key link solved the problems, such as.The drinking water source that current China substantially conforms to standard only accounts for 30% or so, and type is with biology
Based on pollution (69.1%), far above the ratio of chemical contamination.
Countries in the world generally using excrement colibacillus group or ETEC as indicator microoraganism, to reflect microorganisms in water
Pollution situation, but these traditional index are only capable of reflecting the degree of fecal pollution, it is impossible to the definite source of identification pollution, can not
Evaluate the respective contribution rate of different pollution sources.Make it that improvement is remained at present due to lacking the accurate information in fecal pollution source
See the dirty pollution treatment stage, the difficulty and cost of pollution control has been significantly greatly increased.
In recent years, the developed country such as America and Europe has carried out the microorganisms in water based on host specificity biomarker one after another
The research work of source resolution is polluted, and have developed many species-specific primers of the animals such as pig, ox, people, chicken, wild fowl, in time
The accurate information in fecal pollution source is provided timely and effectively to carry out specific aim Sources controlling.However, each in Host Digestion road
Interaction between kind of gut flora is extremely complex, and the otherness of external environment is easily caused the gene of flora in alimentary canal
Change, cause the phenomenon of different regions expression of specific gene difference more universal, spy of the same primer in different regions
The opposite sex there may be very big difference, and the evolution process of environment can not completely be showed by individual gene, therefore should be positive
Carry out the biomarker research of Local Adaptation.So far, in China there is not yet utilizing fluorescent quantitative PCR technique (qPCR)
The research of quantitative judge is carried out to the fecal pollution of the animal such as people, ox, pig and chicken in water body, also without related patented technology.
The content of the invention
It is an object of the invention to provide a kind of microorganisms in water pollution source Methodology of Quantitative Analysis, this method can recognize that low dense
The objective microbe of degree, data are reliable.
The present invention is based on bacteroid host specificity biomarker primer source tracing method, using qPCR technology quantitative analysis environment
The main host source of microorgranic contaminant, comprises the following steps in water body：
(1) primer region property is verified：
It is stored in after gathering different biological fecal specimens, sample collection on regional aim waters periphery in ice chest, in 6h
Send laboratory back to and carry out subsequent treatment；
Using excrement genome DNA extracting reagent kit by suspending, broken, adsorption column the step such as purify from fecal specimens
Genomic DNA is extracted, -20 DEG C of refrigerator storages are placed in；
The genomic DNA extracted using in each biological excrement chooses existing each biological bacteroid specific primer as template
QPCR amplifications are carried out respectively, analyze sensitivity and the specificity of primer；Choose sensitivity and specificity drawing all more than 85%
Thing is used as region-specific primers；
(2) standard curve making：
Enter performing PCR amplification to the genomic DNA of extraction with region-specific primers, the target product after amplification is carried out pure
Change and reclaim, be connected on pMD 19-T Vector carriers, convert DH5 α competent cells, using flat containing ammonia benzyl antibiotic
Plate culture medium picking positive colony, extracts DNA, determines its concentration；
By 10 times of gradient dilutions of plasmid, dilution 10-1、10-2、10-3、10-4、10-5、10-6、10-77 gradients, and as
Template, each gradient sets 3 repetitions as standard items, carries out qPCR amplifications；After reaction terminates, the system of standard curve is carried out
Make；
(3) source resolution is polluted：
Water sample collection is carried out from regional aim waters, is numbered and is stored in ice chest after all samples collection,
The genomic DNA in water sample is extracted in laboratory；QPCR amplifications are carried out using region-specific primers, standard are as a result substituted into bent
Line equation is calculated, and draws the concentration value of each microorganism in target water, according to the concentration value of each microorganism come resolution areas
The pollution sources of target water.
Under preferable case, the qPCR amplification conditions：Amplification system is 20ul：Premix Ex TaqTM II
(2×)10ul；ddH2O, 6.4ul；Each 0.8ul of upstream and downstream primer；DNA profiling 2ul；Amplification program：1. pre-degeneration, 95 DEG C,
30s；2. it is denatured, 95 DEG C, 5s；3. annealing extension, 60 DEG C, 1min；40 circulations.
Under concrete condition, the sensitivity of primer is with specific judgment basis in step (1)：By corresponding to Cq=32.0
Copy number verifies the characteristic of primer as minimum quantitative limit, the Cq expanded when primer<32.0 and melting curve present it is single
It is determined as positive findings during melting peakss, negative findings is determined as during and Cq >=32.0.
Under concrete condition, the judgment basis of step (2) standard curve is：Coefficient correlation (the R of standard curve2) be more than
0.99, credible result；Amplification efficiency (E) determined by slope of standard curve, both coincidence formula E=10(-1/Slope)-
1；If the slope of standard curve of a reaction is -3.32, the amplification efficiency E=100% of the reaction, amplification efficiency (E) exists
It is acceptable between 90%~110%.
The present invention by choose have high specificity, sensitivity high for target area and can quantitative analysis specificity it is raw
Substance markers primer, solves the regional differences of specific biomarker primer, environment water is parsed by qPCR amplification techniques
The quantitative source of microorganism pollution, can recognize that the objective microbe of low concentration, it is adaptable to which the environment water of different quality, is ambient water
The monitoring of body, trace to the source and administer there is provided reliable foundation.
Brief description of the drawings
Fig. 1 is each sampled point indicator microoraganism concentration.
Embodiment
To absolutely prove the characteristic of the present invention and implementing the mode of the present invention, specific embodiment is given below.
First, reagent and method
In specific embodiment, excrement genome DNA extracting reagent kit is given birth to using TIANGEN Biotech (Beijing) Co., Ltd.
The DP328-02 of production, water body genome DNA extracting reagent kit uses Magen bio tech ltd D3145-02, and plasmid is carried
The DP105-03 for taking kit to use TIANGEN Biotech (Beijing) Co., Ltd. to produce, DNA purification kits are given birth to using Tiangeng
Change the DP214-03 of scientific and technological (Beijing) Co., Ltd production.Each specification that can be found in each kit with purification process that extracts enters
OK.Other reagents can be obtained by existing commercial channel.
Standard PCR step：Amplification system is 20 μ L：2×PCR DS Mix 10μL；ddH2O, 7.6 μ L；Upstream and downstream primer
Each 0.8 μ L；The μ L of DNA profiling 0.4.Amplification system is：1. pre-degeneration, 95 DEG C, 5min；2. it is denatured, 95 DEG C, 30s；3. anneal, 60
DEG C, 30s；4. extend, 72 DEG C, 30s, 35 circulations；5. extension, 72 DEG C, 5min eventually.Amplified production is purified.
QPCR steps：Amplification system is 20uL：Premix Ex TaqTM II(2×)10uL；ddH2O,
6.4uL；Each 0.8uL of upstream and downstream primer；DNA profiling 2uL；QPCR amplification programs：1. pre-degeneration, 95 DEG C, 30s；2. it is denatured, 95
DEG C, 5s；3. annealing extension, 60 DEG C, 1min；40 circulations.
2nd, primer region property is verified
2.1 sample collection
Different biological excrement sample is gathered in multiple livestock and poultry farms of the Delta of Pearl River Dong Jiang with Bei Jiang basins
Product, in Guangzhou, hospital gathers human excrement sample.Fecal specimens number amounts to 140 parts, wherein 31 parts of people, 20 parts of chicken, 32 parts of pig, dog 14
Part, 8 parts of rabbit, 28 parts of ox, 7 parts of horse.It is stored in after sample collection in ice chest, sends laboratory in 6h back to and carry out subsequent treatment.
Using excrement genome DNA extracting reagent kit by suspending, broken, adsorption column the step such as purify from fecal specimens
Genomic DNA is extracted, specific steps are carried out to specifications, are placed in -20 DEG C of refrigerator storages.
2.2 characteristic biomarker primers are chosen
Checking examination is carried out from the people source reported, ruminant, pig source and chicken source bacteroid characteristic biomarker primer
Test, specific primer is as shown in table 1.
The characteristic biomarker primer of table 1
The checking of primer is generally carried out using Standard PCR technology, and this method is verified and analyzed using qPCR technologies.
QPCR technologies based on the cycle-index (Cq values) and melting curve that reach threshold value effectively to be analyzed.When the Cq values of each primer
During more than 32.0, it may appear that Cq values error is larger between multiple holes, or even fail to obtain the phenomenons such as amplification.Therefore, in target
In survey region, each primer can verify the copy number corresponding to Cq=32.0 as minimum quantitative limit the characteristic of primer, when
The Cq of primer amplification<Judge when 32.0 and melting curve is determined as positive findings when single melting peakss are presented, and Cq >=32.0
For negative findings.According to this standard, chicken source specific primer 11 have chosen#(qC160F-HU/qBac265R-HU) carry out respectively
Regular-PCR and qPCR amplification experiments, to observe distinct methods to primer sensitivity and specific expressed difference.As a result such as table 2
It is shown.
The result of the PCR and qPCR methods of table 2 to chicken source specific primer
Using 11#The positive sample number that primer is obtained in 20 chicken manure samples by PCR and qPCR methods is respectively 15
Individual and 17, the sensitivity of regular-PCR checking and specificity are respectively less than 80%, and the sensitivity of qPCR checkings and specificity are respectively
For 85% and 90.2%, it is adaptable to goals research region.For illustrating qPCR methods compared with regular-PCR, with higher recall rate,
It is also stronger to the specific amplification of different primers, in primer Qualify Phase and follow-up practical application, with obvious advantage.
The region applicability checking of 2.3 primers
The DNA extracted using in each animal wastes chooses existing people, ruminant, pig and chicken source bacteroid special as template
Specific primer carries out qPCR amplifications.The statistical result and sensitivity and specificity analysis in table 2 and table 3 of each primer amplification.
The amplification of the bacteroid specific primer of table 2
The bacteroid specific primer sensitivity of table 3 is analyzed with specificity
Using people source specific primer (1#~4#) respectively to each fecal specimens carry out qPCR amplifications when, 1#~4#Primer is special
The opposite sex is stronger, but sensitivity removes 1#Reach beyond 100%, it is other poor.Ruminant primer (5#-8#) in, 5#Sensitivity
And specificity is respectively 92.9% and 99.1%, effect is preferable.6#、7#Primer specificity (being 88.2%) is relatively strong, but sensitivity
(being respectively 78.6% and 46.4%) is poor.8#The specificity of primer is relatively strong (97.4%), and sensitivity is only 25%.To pig source
Bacteroid specific primer (9#、10#) checking when, 9#The sensitivity of primer and specificity are 93.8% and 96.4%, the primer
Sensitivity is compared with 10#Primer is higher by 71.9%.Chicken source specific primer 11#With preferable sensitivity (85%) and stronger special
Property (92.3%), respectively than 12#Primer is higher by 25% and 11.2%.Based on the above results, people source specific primer 1#
(qHS601F/qBac725R), ruminant-specific primer 5#(BacB2-590F/Bac708Rm), pig source specific primer 9#
And chicken source specific primer 11 (Bac41F/Bac163R)#(qC160F-HU/qBac265R-HU) have higher sensitivity and
Stronger specificity, it is adaptable to Pearl River Delta region.
3rd, the making of standard curve
Choose the people source specific primer 1 filtered out#(qHS601F/qBac725R), ruminant-specific primer 5#
(BacB2-590F/Bac708Rm), pig source specific primer 9#And chicken source specific primer 11 (Bac41F/Bac163R)#
(qC160F-HU/qBac265R-HU) genomic DNA that primer pair is extracted enters performing PCR amplification, and the target product after amplification is entered
Row purifying is reclaimed, and is connected on pMD 19-T Vector carriers, DH5 α competent cells is converted, using containing ammonia benzyl antibiotic
Plating medium picking positive colony, extract DNA, determine its concentration.
By 10 times of gradient dilutions of plasmid, dilution 10-1、10-2、10-3、10-4、10-5、10-6、10-77 gradients, and as
Template, each gradient sets 3 repetitions as standard items, carries out qPCR amplifications.After reaction terminates, the system of standard curve is carried out
Make, according to standard curve fit formula, concrete outcome such as table 4.
The calibration curve formula of 4 four kinds of primers of table
Coefficient correlation (the R of standard curve2) it is more than 0.99, credible result.Amplification efficiency is that (E) is oblique by standard curve
Rate determines, both coincidence formula E=10(-1/Slope)-1., should if the slope of standard curve of a reaction is -3.32
The amplification efficiency E=100% of reaction.It is generally acknowledged that amplification efficiency (E) is acceptable between 90%~110%.
Plasmid is purer compared to Standard PCR product, more stable, quantitative accurate, it is possible to reduce template error, while can obtain
Get Geng Gao amplification efficiency.Standard PCR product is impure to refer to that PCR primer is not single, can influence follow-up gradient dilution and quantitative
Accuracy.
4th, actual pollution source resolution
Sample collection is carried out to (Z1~Z6) surface water at (G1~G8) at Guangzhou 8, Zhuhai City 6, each sampled point is equal
A water sample is respectively gathered at the left, center, right of river course cross section, altogether 42 parts of water samples.It is numbered and protects after all samples collection
It is stored in ice chest, subsequent experimental is carried out in transporting laboratory in 6h to.Each sampled point is as shown in table 5.
Each sampling point distributions table of table 5
| Place city | Sampled point | Referred to as |
| Guangzhou | Increase Jiangkou | G1 |
| Guangzhou | Tatun | G2 |
| Guangzhou | Shawan | G3 |
| Guangzhou | Dong Long | G4 |
| Guangzhou | Liang Kou towns | G5 |
| Guangzhou | Hot spring town | G6 |
| Guangzhou | Flow the small stream territory village | G7 |
| Guangzhou | Renhe Town | G8 |
| Zhuhai | Xiang Zhou gulfs | Z1 |
| Zhuhai | Jilin University's sewage draining exit | Z2 |
| Zhuhai | Stone angle is bright | Z3 |
| Zhuhai | Wood is beach | Z4 |
| Zhuhai | Chicken crows door | Z5 |
| Zhuhai | Chicken crows a mouthful marine outfall | Z6 |
Measure every part of water sample 100mL to filter by vacuum filter using 0.22 μm of filter membrane, thalline is stayed on filter membrane,
And the thallus DNA stayed on filter membrane is extracted according to water body DNA extraction kit specification.Using people source specific primer 1#
(qHS601F/qBac725R), ruminant-specific primer 5#(BacB2-590F/Bac708Rm), pig source specific primer 9#
And chicken source specific primer 11 (Bac41F/Bac163R)#(qC160F-HU/qBac265R-HU) qPCR amplifications are carried out, as a result generation
Enter calibration curve equation formula to be calculated, draw the concentration value of each microorganism in target water, test result is as shown in Figure 1.
The characteristic of bacteroid obligate anaerobic can also make its rapid decay in water body, therefore bacteroid is usual as indicator bacteria
For assessing recent pollution.But in urban water-body, because human excrement may be from undressed sanitary sewage, municipal pipeline
Sewage overflow or the number of ways such as sewage treatment plant so that people source specificity bacteroid can maintain higher concentration level.
From Fig. 1 it is observed that qHB concentration is averagely higher by 1~2 order of magnitude compared with qRB, 2~4 orders of magnitude are higher by compared with qCB, illustrate each
Water body is mainly polluted more serious by human excrement, and this also mainly receives the actual feelings of sanitary sewage and municipal wastewater with urban water-body
Condition is consistent.And qRB, qCB that each sampled point is detected also indicate that water body while being polluted by ruminant, poultry dung.But
Found during actual samples, in addition to having part plant and arable land near G5, G6 and Z4, Z5, Z6, where remaining sampled point
River both sides is mainly shopping centre, residential block, and this qRB, qCB illustrated in water body may come for the migration of upstream pollution sources, with
Contamination characteristics are consistent on the spot.
The present invention by choose identify Pearl River Delta area with high specificity, sensitivity it is high and can quantitative analysis people
Source, Niu Yuan, pig source and chicken source specific biomarker primer, solve the regional differences of specific biomarker primer, pass through
QPCR amplification techniques can recognize that the objective microbe of low concentration come the quantitative source of microorganism pollution for parsing environment water, it is adaptable to
The environment water of different quality.
Claims (7)
1. a kind of microorganisms in water pollution source Methodology of Quantitative Analysis, comprises the following steps：
(1) primer region property is verified：
It is stored in after gathering different biological fecal specimens, sample collection on regional aim waters periphery in ice chest, sends experiment back to
Room carries out subsequent treatment；
Base is extracted from fecal specimens by suspending, crushing, adsorb column purification step using excrement genome DNA extracting reagent kit
Because of a group DNA, refrigerator low-temperature storage is placed in；
The genomic DNA extracted using in each biological excrement chooses existing each biological bacteroid specific primer difference as template
QPCR amplifications are carried out, sensitivity and the specificity of primer is analyzed；Choose the higher primer of sensitivity and specificity special as region
Specific primer；
(2) standard curve making：
Enter performing PCR amplification to the genomic DNA of extraction with region-specific primers, the target product after amplification is purified back
Receive, be connected on pMD 19-T Vector carriers, convert DH5 α competent cells, trained using the flat board containing ammonia benzyl antibiotic
Base picking positive colony is supported, DNA is extracted, determines its concentration；
By plasmid gradient dilution, and as template, qPCR amplifications are carried out；After reaction terminates, the making of standard curve is carried out；
(3) source resolution is polluted：
Water sample collection is carried out from regional aim waters, is numbered and is stored in ice chest after all samples collection, in experiment
The genomic DNA in water sample is extracted in room；QPCR amplifications are carried out using region-specific primers, as a result through calibration curve formula
Calculated, draw the concentration value of each microorganism in target water, according to the concentration value of each microorganism come resolution areas target water
The pollution sources in domain.
2. microorganisms in water pollution source Methodology of Quantitative Analysis according to claim 1, wherein the qPCR amplification conditions：
Amplification system is 20ul：Premix Ex TaqTM II(2×)10ul；ddH2O, 6.4ul；Upstream and downstream primer is each
0.8ul；DNA profiling 2ul；Amplification program：1. pre-degeneration, 95 DEG C, 30s；2. it is denatured, 95 DEG C, 5s；3. annealing extension, 60 DEG C,
1min；40 circulations.
3. the sensitivity of primer in microorganisms in water pollution source Methodology of Quantitative Analysis according to claim 1, step (1)
It is with specific judgment basis：Copy number corresponding to Cq=32.0 is verified into the characteristic of primer as minimum quantitative limit, when
The Cq of primer amplification<Judge when 32.0 and melting curve is determined as positive findings when single melting peakss are presented, and Cq >=32.0
For negative findings.
4. microorganisms in water pollution source Methodology of Quantitative Analysis according to claim 1, step (2) standard curve is sentenced
Determining foundation is：Coefficient correlation (the R of standard curve2) it is more than 0.99, credible result；Amplification efficiency (E) is oblique by standard curve
Rate determines, both coincidence formula E=10(-1/Slope)-1；, should if the slope of standard curve of a reaction is -3.32
The amplification efficiency E=100% of reaction, amplification efficiency (E) is acceptable between 90%~110%.
5. in microorganisms in water pollution source Methodology of Quantitative Analysis according to claim 1, step (1), choose sensitivity
Region-specific primers are used as with primer of the specificity all more than 85%.
6. plasmid is pressed 10 times in microorganisms in water pollution source Methodology of Quantitative Analysis according to claim 1, step (2)
Gradient dilution, dilution 10-1、10-2、10-3、10-4、10-5、10-6、10-77 gradients, each gradient sets 3 weights as standard items
It is multiple.
7. the microorganisms in water pollution source Methodology of Quantitative Analysis according to one of claim 1-6, the region is China
Pearl River Delta area.
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| CN111549105A (en) * | 2020-04-01 | 2020-08-18 | 生态环境部华南环境科学研究所 | Evaluation method for identifying applicability of specific marker in area |
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| CN113444818B (en) * | 2021-06-04 | 2023-03-31 | 生态环境部华南环境科学研究所 | Microbial pollution source analysis method for effectively eliminating false positive reaction |
| CN115798590A (en) * | 2022-12-26 | 2023-03-14 | 上海亿康医学检验所有限公司 | Sample tracing method, sample storage vessel, equipment and readable storage medium |
| CN117512149A (en) * | 2023-12-01 | 2024-02-06 | 北京师范大学 | Microbial gene fingerprint spectrum for identifying pollution of shrimp and crab culture sources and application thereof |
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