CN104846113A - PCR kit capable of simultaneously detecting fecal pollution of dogs, pigs and chickens in water and detection method thereof - Google Patents
PCR kit capable of simultaneously detecting fecal pollution of dogs, pigs and chickens in water and detection method thereof Download PDFInfo
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- 230000002550 fecal effect Effects 0.000 title claims abstract description 56
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 35
- 238000001514 detection method Methods 0.000 title claims abstract description 22
- 238000009004 PCR Kit Methods 0.000 title claims abstract description 9
- 241000282472 Canis lupus familiaris Species 0.000 title abstract 5
- 241000282887 Suidae Species 0.000 title abstract 5
- 235000013330 chicken meat Nutrition 0.000 title abstract 5
- 238000000034 method Methods 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 11
- 102000004190 Enzymes Human genes 0.000 claims abstract description 11
- 238000012408 PCR amplification Methods 0.000 claims abstract description 10
- 238000000246 agarose gel electrophoresis Methods 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 230000004087 circulation Effects 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 230000008021 deposition Effects 0.000 claims description 4
- 238000001502 gel electrophoresis Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 108020004414 DNA Proteins 0.000 description 32
- 210000003608 fece Anatomy 0.000 description 23
- 239000000243 solution Substances 0.000 description 11
- 238000001962 electrophoresis Methods 0.000 description 9
- 108020005196 Mitochondrial DNA Proteins 0.000 description 7
- 239000010871 livestock manure Substances 0.000 description 7
- 239000002689 soil Substances 0.000 description 7
- 239000012634 fragment Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 241000272525 Anas platyrhynchos Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003911 water pollution Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 102100025287 Cytochrome b Human genes 0.000 description 1
- 108010075028 Cytochromes b Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
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- 244000144977 poultry Species 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
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- 239000001226 triphosphate Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
The invention discloses a PCR kit capable of simultaneously detecting fecal pollution of dogs, pigs and chickens in water and a detection method thereof, belonging to the field of detection of water body fecal pollution. The kit capable of simultaneously detecting fecal pollution of dogs, pigs and chickens in water comprises a primer, dNTP, PCR buffer, a Mg<2+> solution and ddH20. The method for detecting the water body fecal pollution by utilizing the kit comprises the following steps: extracting DNA of a to-be-detected water sample, taking the DNA as a template, performing PCR amplification by utilizing the primer, dNTP, PCR buffer, Taq enzymes, Mg<2+> solution and ddH20 provided in the kit, performing agarose gel electrophoresis on the PCR product, observing under an ultraviolet lamp, wherein if stripes of 390bp, 490bp and 783bp occur, fecal pollution of dogs, pigs and chickens respectively exists in the water. According to the kit, the fecal pollution of dogs, pigs and chickens, which independently exists or exists in a random combination manner in water can be accurately detected, more than three types of fecal pollution can be simultaneously detected at most, the detection time and cost can be greatly saved when a fecal pollution source is checked, and the method has good application prospects.
Description
Technical field
The present invention relates to water pollution detection field, more particularly, relate to a kind of PCR kit and the detection method thereof that detect dog, pig, chicken fecal pollution in water simultaneously.
Background technology
In recent years, along with the growth of demographic and economic, water pollution situation is on the rise, and wherein fecal pollution is particularly outstanding.Fecal pollution thing not only can increase water body nitrogen and phosphorus content after entering water body, cause body eutrophication, and the pathogenic bacterium that may exist in ight soil can be brought into, diseases induced water body is propagated (Baldursson S and Karanis P.Waterborne transmission ofprotozoan parasites:Review of worldwide outbreaks ?An update 2004 ?2010.Water Research 2011; 45 (20): 6603 ?6614.).Therefore the control of water body fecal pollution is carried out very urgent.
The potential pollution source of fecal pollution is of a great variety, the excrement of the common species such as the pig in livestock and poultry breeding industry, chicken, owing to lacking suitable process, usually directly be discharged in environment, cause water body fecal pollution, in addition, along with the quantity of pet (especially pet dog) gets more and more, its excrement produced also becomes one of source of water body fecal pollution.In point source, the simultaneous situation of pollution of area source, lack the accurate grasp to fecal pollution source and location, make control function rest on " seeing dirty pollution treatment " stage, add the cost of Pollution abatement to a certain extent.How to find and distinguish and fast, accurately locate the fecal pollution source in water body, becoming the key of dealing with problems.
Tradition fecal pollution detection method judges water body fecal pollution situation by detecting fecal pollution indicator (as intestinal bacteria, faecalis etc.) in water, length consuming time and cannot distinguish source of pollution.By detecting water body host specificity microorganism or chemical marker, the object that fecal pollution is traced to the source can be reached, but some existing microorganisms and chemical marker specificity not high, often cause the generation of false positive results.Exist in a large number from the Mitochondrial DNA of the intestinal cell that comes off in ight soil, because the Mitochondrial DNA of different plant species is different, wherein there is a large amount of mononucleotide polymorphic site in specific fragment, Mitochondrial DNA is made to have species specificity, can reach by the mitochondria DNA fragment in fecal pollution water body the object (Caldwell J, PaymentP and Villemur R (2011) Mitochondrial DNA as Source Tracking Markers of Fecal Contamination.) that fecal pollution traces to the source by detecting.
The detection that the fast development of the Protocols in Molecular Biology based on PCR is water body fecal pollution provides strong instrument.But the fecal pollution source tracing method of at present conventional PCR-based means can only detect separately source of pollution usually (Caldwell JMand Levine JF.Domestic wastewater influent profiling using mitochondrial real ?time PCR for sourcetracking animal contamination.Journal of microbiological methods 2009; 77 (1): 17 ?22.Schill WBand Mathes MV.Real ?time PCR detection and quantification of mine potential sources of fecalcontamination by analysis of mitochondrial cytochrome b targets.Environmental science & technology 2008; 42 (14): 5229 ?5234.).Not yet openly can distinguish the detection method of multiple source of pollution at present.The present invention is by the mtdna sequence of comparison different plant species, have found dog, pig, specific chicken DNA fragmentation, and devise Auele Specific Primer for different fragments, and the expanding fragment length of different primers has certain gap, district pig, dog, chicken three kinds of fecal pollution sources in a PCR reaction system are made to become possibility.
Summary of the invention
The problem that 1, will solve
The PCR method of current detection water body fecal pollution is normally investigated one by one to potential pollution source, consuming time for a long time and cost is high.For this problem, the invention provides and a kind ofly can detect dog in water body, pig, the PCR kit of chicken fecal pollution and detection method thereof simultaneously, can reach fast, accurate investigation or detect the object in water body fecal pollution source.
2, technical scheme
Object of the present invention is achieved through the following technical solutions
Detect PCR method and the test kit of dog, pig, chicken fecal pollution in water simultaneously, comprise primer, Taq enzyme, dNTP, PCR buffer, Mg
2+solution and ddH
20, wherein primer comprises:
Primer 1:5 ’ ?CTGTGCTATGTCAGTATCTCCAG ?3 ';
Primer 2: 5 ’ ?GGCTGATTAGTCATTAGTCCATCG ?3 ';
Primer 3:5 ’ ?CTACTCATACCCAGCAAGCC ?3 ';
Primer 4:5 ’ ?TAGGAATTAATAGGGCGGGTG ?3 ';
Primer 5:5 ’ ?CACATGTTATCTGCACCAGC ?3 ';
Primer 6:5 ’ ?GTTAAGGGTACGAGTTTGTCG ?3 '.
Further, described PCR buffer is 10 × PCR buffer, dNTP concentration is each 2.5mmol/L, and Taq enzyme concentration is 5U/ul, Mg
2+strength of solution is 25mmol/L.。
Adopt the method for the detection water body fecal pollution of above-mentioned PCR kit, concrete steps are as follows: extract water sample DNA to be measured, with water sample DNA to be measured for template, utilize the primer 1 that provides in mentioned reagent box ?6, dNTP, PCR buffer, Mg
2+solution and ddH
20 carries out pcr amplification, and PCR primer exists situation by observing band after agarose gel electrophoresis under ultraviolet lamp, judges water body fecal pollution situation with this.
Concrete, the method utilizing mentioned reagent box to detect water body fecal pollution is:
(1) water sample DNA is extracted;
(2) with water sample DNA for template, utilize provide in test kit primer, dNTP, PCR buffer, Mg
2+solution and ddH
20 carries out pcr amplification, and reaction system is: 10 × PCR buffer of 5ul; 5ul dNTP; 0.25ul Taq enzyme; 4ul Mg
2+; The mix primer 1 of 3ul ?6, wherein the concentration of each primer is 10umol/L; 4ul template DNA; Sterilizing ddH
2o complements to 50ul; Reaction conditions is 95 DEG C, denaturation 5min; 95 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 45s, 40 circulations; 72 DEG C of 7min, 4 DEG C of preservations;
(3) detect pcr amplification product: 5ul PCR primer is carried out 1% agarose gel electrophoresis, 1 × TAE is as electrophoresis apparatus, and EB dyes, and deposition condition is voltage 80V, electric current 400mA, time 25min.(4) electrophoresis observes gel after terminating under ultraviolet lamp; The band of 390bp, 490bp and 783bp that gel occurs, respectively corresponding instruction dog, pig, chicken fecal pollution.
Further, this test kit can detect dog that Individual existence in water body or random combine exist, pig, chicken fecal pollution, above three kinds of fecal pollutions can be detected at most simultaneously.
3, beneficial effect
Compared to prior art, the invention has the advantages that and can detect dog, pig, chicken three kinds of fecal pollutions in a PCR reaction simultaneously, the dog of Individual existence or random combine existence in water body, pig, chicken fecal pollution can be detected, for the water body that fecal pollution situation is more complicated, the object getting rid of or determine fecal pollution source as early as possible can be reached, save time and cost.
The Contaminative markers thing that the present invention chooses is the Mitochondrial DNA of different animals, detection method is made to have good specificity, it is more clear that the band that during electrophoresis detection, different animals ight soil is corresponding is separated, and accurately can judge the ownership of different band, reduces the erroneous judgement detecting sample.
Accompanying drawing explanation
Fig. 1: the present invention is to the electrophoresis result figure of dog, pig, chicken manure just independent and random two kinds of combine detection.
Fig. 2: the electrophoresis result figure that the present invention detects dog, pig, chicken mixing ight soil.
Fig. 3: specific assay electrophoresis result figure of the present invention.
Fig. 4: sensitivity assays electrophoresis result figure of the present invention.
Embodiment
Embodiment 1: to dog, pig, chicken manure just independent or random two kinds of combine detection
Detect PCR method and the test kit of dog, pig, chicken fecal pollution in water body simultaneously, comprise primer, Taq enzyme, dNTP, PCRbuffer, Mg
2+solution and ddH
20, wherein primer comprises:
Primer 1:5 ’ ?CTGTGCTATGTCAGTATCTCCAG ?3 '
Primer 2: 5 ’ ?GGCTGATTAGTCATTAGTCCATCG ?3 '
Primer 3:5 ’ ?CTACTCATACCCAGCAAGCC ?3 '
Primer 4:5 ’ ?TAGGAATTAATAGGGCGGGTG ?3 '
Primer 5:5 ’ ?CACATGTTATCTGCACCAGC ?3 '
Primer 6:5 ’ ?GTTAAGGGTACGAGTTTGTCG ?3 '
Above-mentioned PCR primer customizes synthesis by Sangon Biotech (Shanghai) Co., Ltd. and obtains.
Above-mentioned ddH
2o is sterilizing distilled water.Taq enzyme concentration is 5U/ul.Mg
2+the concentration of solution is 25mmol/L, is MgCl
2solution.The concentration of dNTP is 2.5mmol/L.PCR buffer is 10 × PCR damping fluid (buying in Takara, Code No.RR001A)
It is QIAamp DNA Stool Mini Kit that faeces DNA extracts test kit, for extracting different faeces DNA.
Gather fresh dog, pig, chicken manure just, extract DNA with 0.2 gram of ight soil respectively, extract DNA by after two kinds of ight soil mixing at random in addition.Respectively with separately or mixing faeces DNA carry out pcr amplification for template, PCR reaction system is: 10 × PCR damping fluid (PCR buffer, Takara) of 5ul; 5ul deoxyribonucleoside triphosphate (dNTP); 0.25ul Taq enzyme; The 25mmol/L Mg of 4ul
2+solution; 3ul concentration is the mix primer of 10umol/L; 4ul template DNA; Sterilizing ddH
2o complements to 50ul; Reaction conditions is 95 DEG C, denaturation 5min; 95 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 45s, 40 circulations; 72 DEG C of 7min, 4 DEG C of preservations.
After PCR reaction terminates, the PCR primer of getting 5ul tri-kinds of faeces DNAs respectively carries out 1% agarose gel electrophoresis, and deposition condition is voltage 80V, electric current 400mA, time 25min.
After electrophoresis terminates, observe under gel being placed on ultraviolet lamp, glue figure as shown in Figure 1.Respectively with dog, pig, chicken faeces DNA is occurred in the PCR primer of template that length is 390bp, 490bp, with the band of 783bp, dog, occur that length is the band of 390bp and 490bp in the PCR primer of pig mixing faeces DNA, pig, occur that length is the band of 490bp and 783bp in the PCR primer of chicken mixing faeces DNA, dog, occur that length is the band of 390bp and 783bp in the PCR primer of chicken mixing faeces DNA, illustrate that mix primer in the present invention and PCR method used can accurately detect single dog, pig, chicken fecal pollution and random two kinds of mixing fecal pollutions.
Embodiment 2: simultaneously detect dog, pig, chicken manure just
Take 0.05 gram of dog, pig, chicken manure respectively just, extract DNA after mixing, with this hybrid dna for template carries out pcr amplification, PCR reaction system is: 10 × PCR buffer of 5ul; 5ul dNTP; 0.25ul Taq enzyme; The 25mmol/L Mg of 4ul
2+solution; The mix primer 1 of 3ul ?6, wherein the concentration of each primer is; 4ul template DNA; Sterilizing ddH
2o complements to 50ul; Reaction conditions is 95 DEG C, denaturation 5min; 95 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 45s, 40 circulations; 72 DEG C of 7min, 4 DEG C of preservations.
After PCR reaction terminates, the PCR primer of getting 5ul tri-kinds of faeces DNAs respectively carries out 1% agarose gel electrophoresis, and deposition condition is voltage 80V, electric current 400mA, time 25min.
After electrophoresis terminates, observe under gel being placed on ultraviolet lamp, glue figure as shown in Figure 2.With ddH
20 is without any band in the negative control of template, mixing faeces DNA is occurred in the PCR primer of template that length is the band of 390bp, 490bp and 783bp, illustrates that test kit in the present invention and PCR method used can detect dog, pig, chicken four kinds of fecal pollutions simultaneously.
Embodiment 3: test kit specificity verification
People, ox, sheep, the specificity of duck faeces DNA to test kit in the present invention and PCR method is utilized to verify.Gather Freshman, ox, sheep, duck ight soil extraction DNA, with these four kinds of faeces DNAs for template carries out pcr amplification, mix faeces DNA for positive control, ddH with dog, pig, chicken simultaneously
20 is negative control, and PCR reaction system and condition are with embodiment 2, and Gel electrophoresis conditions is with embodiment 2, and result as shown in Figure 3.Negative control and people, ox, sheep, duck faeces DNA are all without any band in the PCR primer of template, occur in positive control that length is the band of 390bp, 490bp and 783bp, illustrate test kit in the present invention and PCR method specificity used good, there will not be false positive results.
Embodiment 4: test kit susceptibility is verified
Take 0.2 gram of dog, pig, chicken manure respectively and just extract DNA, utilize that NanoDrop 2000 records dog, pig, chicken faeces DNA concentration are respectively 3148.6ng/ul, 96.5ng/ul, 35ng/ul.Carry out 4 times of dilutions by after above-mentioned DNA equal-volume mixing, namely dilute 4
1, 4
2, 4
3, 4
4, 4
5, 4
6doubly, then with each dilution DNA solution for template, carry out pcr amplification, PCR reaction system and condition are with embodiment 2, and Gel electrophoresis conditions is with embodiment 2, and result is as shown in Figure 4.Extension rate is 4
1?4
5pCR primer all occurred that length is respectively the positive band of 390bp, 490bp, 783bp, extension rate is greater than 4
5time there is not band, illustrate test kit in the present invention and the corresponding dog of PCR method, pig, chicken manure detectability be respectively 0.145,0.094,0034ng/ul faeces DNA.
Claims (6)
1. detect a PCR kit for dog, pig, chicken fecal pollution in water simultaneously, comprise primer, dNTP, PCR buffer, Taq enzyme, Mg
2+solution and ddH
20, it is characterized in that: described primer is mix primer, comprising:
Primer 1:5 ’ ?CTGTGCTATGTCAGTATCTCCAG ?3 ';
Primer 2: 5 ’ ?GGCTGATTAGTCATTAGTCCATCG ?3 ';
Primer 3:5 ’ ?CTACTCATACCCAGCAAGCC ?3 ';
Primer 4:5 ’ ?TAGGAATTAATAGGGCGGGTG ?3 ';
Primer 5:5 ’ ?CACATGTTATCTGCACCAGC ?3 ';
Primer 6:5 ’ ?GTTAAGGGTACGAGTTTGTCG ?3 '.
2. the PCR kit simultaneously detecting dog, pig, chicken fecal pollution in water according to claim 1, it is characterized in that, described PCR buffer is 10 × PCR buffer, and described dNTP concentration is each 2.5mmol/L, and described Taq enzyme concentration is 5U/ul, described Mg
2+strength of solution is 25mmol/L.
3. adopt the method for the detection water body fecal pollution of the PCR kit according to any one of claim 1 or 2, it is characterized in that, concrete steps are as follows: extract water sample DNA to be measured, with water sample DNA to be measured for template, utilize the primer 1 that provides in test kit ?6, dNTP, PCR buffer, Mg
2+solution and ddH
20 carries out pcr amplification, and PCR primer exists situation by observing band after agarose gel electrophoresis under ultraviolet lamp, judges water body fecal pollution situation with this.
4. the method for detection water body fecal pollution according to claim 3, is characterized in that, described pcr amplification reaction carries 10 × PCR buffer that system is 5ul; 5ul dNTP; 0.25ul Taq enzyme; 4ul Mg
2+; The mix primer 1 of 3ul ?6, wherein the concentration of each primer is; 4ul template DNA; Sterilizing ddH
2o complements to 50ul; Reaction conditions is 95 DEG C, denaturation 5min; 95 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 45s, 40 circulations; 72 DEG C of 7min, 4 DEG C of preservations.
5. the method for detection water body fecal pollution according to claim 3, is characterized in that, described gel electrophoresis is 1% agarose gel electrophoresis, and deposition condition is: voltage 120V, electric current 440mA, time 25min.
6. the method for detection water body fecal pollution according to claim 3, it is characterized in that, judge that the method for water body fecal pollution situation is: if there is the band of 390bp, 490bp and 783bp after gel electrophoresis, then there is dog, pig, chicken fecal pollution in corresponding instruction in water respectively.
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Cited By (7)
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| CN105603108A (en) * | 2016-03-22 | 2016-05-25 | 重庆大学 | Kit for dog feces pollution detection as well as preparation and application thereof |
| CN105648097A (en) * | 2016-03-22 | 2016-06-08 | 重庆大学 | Dog feces pollution detection kit and preparation and application thereof |
| CN106434921A (en) * | 2016-09-29 | 2017-02-22 | 中国科学院城市环境研究所 | Microbial source tracking molecular marker and high-throughput detection method thereof for detecting multiple fecal pollution sources |
| CN110484637A (en) * | 2019-09-19 | 2019-11-22 | 南京大学 | Primer, kit and the high-throughput source tracing method of a kind of water body fecal pollution detection |
| CN111321236A (en) * | 2020-03-23 | 2020-06-23 | 中国科学院大学 | Microorganism tracing method for chicken manure pollution |
| CN111440883A (en) * | 2020-04-07 | 2020-07-24 | 中国科学院大学 | Pig manure polluted microorganism tracing method |
| US11744725B2 (en) | 2016-08-12 | 2023-09-05 | Coloplast A/S | Ostomy appliance |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603108A (en) * | 2016-03-22 | 2016-05-25 | 重庆大学 | Kit for dog feces pollution detection as well as preparation and application thereof |
| CN105648097A (en) * | 2016-03-22 | 2016-06-08 | 重庆大学 | Dog feces pollution detection kit and preparation and application thereof |
| CN105648097B (en) * | 2016-03-22 | 2019-03-15 | 重庆大学 | A kind of dog fecal pollution detection kit and its preparation and application |
| US11744725B2 (en) | 2016-08-12 | 2023-09-05 | Coloplast A/S | Ostomy appliance |
| CN106434921A (en) * | 2016-09-29 | 2017-02-22 | 中国科学院城市环境研究所 | Microbial source tracking molecular marker and high-throughput detection method thereof for detecting multiple fecal pollution sources |
| CN110484637A (en) * | 2019-09-19 | 2019-11-22 | 南京大学 | Primer, kit and the high-throughput source tracing method of a kind of water body fecal pollution detection |
| WO2021051622A1 (en) * | 2019-09-19 | 2021-03-25 | 南京大学 | Primers, kit, and high-throughput traceability method for detecting fecal contamination in water bodies |
| CN111321236A (en) * | 2020-03-23 | 2020-06-23 | 中国科学院大学 | Microorganism tracing method for chicken manure pollution |
| CN111440883A (en) * | 2020-04-07 | 2020-07-24 | 中国科学院大学 | Pig manure polluted microorganism tracing method |
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