CN103451295A - Method for biologically assessing quality of polluted water - Google Patents
Method for biologically assessing quality of polluted water Download PDFInfo
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- CN103451295A CN103451295A CN2013104030586A CN201310403058A CN103451295A CN 103451295 A CN103451295 A CN 103451295A CN 2013104030586 A CN2013104030586 A CN 2013104030586A CN 201310403058 A CN201310403058 A CN 201310403058A CN 103451295 A CN103451295 A CN 103451295A
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Abstract
The invention discloses a method for biologically assessing the quality of polluted water. The method is mainly used for assessing the potential ecotoxicological effect existing after water is polluted by utilizing an expression pattern of an hsp70 gene of procambarus clarkia. The hsp70 gene is obtained from liver tissues of procambarus clarkia through cloning for the first time by adopting the method. Industrial sewage stress experiment results show that up-regulated expression of the hsp70 gene of procambarus clarkia is directly correlated with sewage stress, and the degree of the up-regulated expression quantity of the hsp70 gene is related to the degree of water pollution. Therefore, a comprehensive and scientific method is provided for assessing the quality of water by utilizing the biotechnical means, and has a relatively wide application space in water environment protection.
Description
Technical field
The present invention relates to the biological method that a kind of Procambius clarkii hsp70 gene expression pattern is estimated for Water quality, belong to the field of Environmental Biotechnology.
Background technology
Along with the economic sustained and rapid development of China, the urbanization progress in China rural area is keeping the industrialization degree in the good impetus, especially a rural area, small towns to improve constantly always, makes diversified enterprise enter among the common people's daily life.So just between enterprise's pursuit economic interests and the people's daily life, set up a kind of the contact, the essence of this contact is trial of strength and the balance between rapid economic development and environment sustainable development in fact.At present, the Chinese government has clearly proposed to using to set up friendly environment society and has grabbed as a long-term and unremitting task in " 12 " planning, strives for the Sustainable development of Chinese society.Wherein, the benign development of water surrounding is directly connected to the people's healthy problem.In recent years, the event that water pollution causes pernicious consequence frequently occurs in China, for example, the children blood lead event in Huaining, Anhui, Guangzhou cadmium exceed standard rice event, Fujian Zijin Mining effluent seepage event etc., these events have its source in our daily life directly or the water source of indirect correlation contaminated.In the face of these problems, we need a kind of novel method that can the thoroughly evaluating Water quality, require this method can reflect the potential ecological toxicological effect existed after water pollution simultaneously, can clearly tell which type of biology consequence this pollution of the people can cause.
At present, environmental administrations at different levels are when being detected for Water quality, and what usually adopt is chemical measure.The chemical detection method is mainly by usining known reference material as reference, determines kind and the concentration of pollutent unknown in detected water sample by the mensuration of precision instrument.Yet, from the biology angle, there is the cumulative problem of a larger effect at this chemical measure, if a detected contaminated water sample is by the result of chemical measure, the content of a plurality of pollutents can be detected, but the content of these pollutents does not surpass national standard, illustrate that this pollution can ignore or think very slight pollution.But, from biological angle, all polluting effects can extensive connection in organism, in water body, all pollutents that do not exceed standard are to add up for the influence of organism.Therefore, the potential ecological toxicological effect that chemical measure can not a kind of water pollution of thoroughly evaluating meeting causes, simultaneously, we need to set up the novel method of a set of perfect biological assessment Water quality.
Procambius clarkii is commonly called as freshwater crayfish, and it is the model animals of Crustacean, at south China and the north, cultivation is arranged, and for the result of study of biological test, has certain indication and role of delegate.Because Procambius clarkii is convenient to obtain, be easy to raise, there is again the meaning of representative, so be suitable as very much the indicator organism of water body environment quality simultaneously.We can be subject to after water body environment coerces stimulation by Procambius clarkii, detect the expression of certain gene in the Procambius clarkii body, reflect this ecological toxicological effect of coercing, thereby indirectly reflect the quality of water body.
The full name of Procambius clarkii hsp70 gene is the emergent protein 70 gene of Procambius clarkii; this gene is the marker gene that animal body reply external environment is coerced stimulation; when animal body is subject to external environment and coerces; can alleviate this bad physiology consequence caused of coercing by the expression amount of regulating the hsp70 gene; carry out autoprotection; be the equal of a kind of immune defence mechanism, and the expression amount of hsp70 gene is raised in this adjusting often.Like this we can the expression amount of hsp70 gene and external environment coerce between degree set up a kind of associated, thereby the indicator using the hsp70 gene as the indication Water quality.Simultaneously, Procambius clarkii 18S rRNA gene is the gene (house-keeping gene) of a constant expression of animal body, and the expression amount of this gene can not be affected because of extraneous stimulation.Therefore, by the expression by Procambius clarkii hsp70 gene and 18S rRNA gene, compare, just can obtain the relative expression quantity of the Procambius clarkii hsp70 gene under the environment-stress hormesis, can further reflect the pollution condition of water body environment by the variation tendency of relative expression quantity.
For a long time; detection for the polluted water weight; environmental protection department adopts the method for chemical detection more; this method exists detection means single; can not reflect the potential ecological toxicological effect that polluted water body exists, we need to set up the novel method of a set of perfect biological assessment Water quality comprehensively.
Summary of the invention
The object of the present invention is to provide a kind of hsp70 of utilization gene expression pattern to carry out the biological method of qualitative evaluation water pollution degree, and this method can reflect the potential ecological toxicological effect that water pollution exists.
Technical solution:
Concrete grammar of the present invention is as follows:
1) precuring of Procambius clarkii: buy the Procambius clarkii lived from local fish market, requiring the body weight of shrimp is 20g-30g, the quantity of shrimp is 90, get back to testing laboratory and be divided into 3 groups, every group 30, with raising 1 day-2 days through the tap water of sterilising treatment, allow its adequacy test environment in aquarium, obtain the precuring Procambius clarkii;
2) pre-treatment of detected Wastewater Sample: will gather the Wastewater Sample come and be diluted, extension rate is generally between 5 times-10 times, and is equally divided into 3 parts, obtains 3 groups of water samples after diluting;
3) Procambius clarkii coerce irritant test: 3 groups of precuring Procambius clarkiis in step (1) are put among 3 groups of water samples after the dilution of step (2), simultaneously, every group reserves 3 Procambius clarkiis as the normal control sample, other 27 Procambius clarkiis start to carry out sewage and coerce irritant test, coerce the stimulating course requirement and carry out in aquarium, obtain the Procambius clarkii of coercing stimulation;
4) extraction of the total RNA of Procambius clarkii: the Procambius clarkii of coercing stimulation in step (3) is usingd and hour sampled as time of day, the time point of sampling is respectively coerces the 6h stimulated after starting, 12h, 24h, 48h, 72h, 96h, on each time point, require to coerce the Procambius clarkii of getting 3 work the stimulation test group from every group, gather the liver organization of Procambius clarkii under the aseptic condition of Bechtop, the sampling amount that requires every Procambius clarkii liver is 20mg-30mg, after mixing, the liver organization of 3 shrimps carries out the extraction of total RNA, obtain the Procambius clarkii liver total RNA sample that each sampling time point is corresponding,
5) reverse transcription of Procambius clarkii cDNA is synthetic: the corresponding Procambius clarkii liver total RNA sample by each sampling time point in step (4), the single stage method RT-PCR amplification kit (M-MuLV) that utilizing Shanghai to give birth to work biotechnology company limited provides carries out the reverse transcription of cDNA and synthesizes, and concrete treatment process is: in the Eppendorf tube of nuclease free, add Oligo (dT)
20(50 μ M) 1 μ L, total RNA sample liquid 2 μ L, 10mM dNTP mixture 1 μ L, RNase-free ddH
2o8 μ L, and, after this mixture is placed in to 65 ℃ of water-bath heating 5min-10min, be placed in rapidly cooling 5min-10min on ice bath, after of short duration low-speed centrifugal, add 5 * the first chains to synthesize damping fluid 4 μ L, 0.1M DTT2 μ L, RNaseOUT
tM nucleic acid inhibitor 1 μ L, and, in centrifuge tube, gently various compositions are mixed, hatch 2min-3min in 37 ℃ of water-baths, then add at ambient temperature the reversed transcriptive enzyme of 1 μ L, piping and druming mixes lightly, in 37 ℃ of water-baths, hatches 5min-10min, finally under 70 ℃ of water bath condition, heating 15min-20min, with termination reaction, obtains the Procambius clarkii liver organization cDNA sample that each sampling time point is corresponding;
6) Real-Time Fluorescent Quantitative PCR Technique is determined the expression pattern of Procambius clarkii hsp70 gene: the Procambius clarkii liver organization cDNA sample that each sampling time point in step (5) is corresponding is as template, simultaneously, the complete nucleotide sequence design real-time fluorescence quantitative PCR of the Procambius clarkii hsp70 gene obtained according to we clone is tested needed primer hsp70-qRT-F:5`-caccagatgaaggagttg-3` and hsp70-qRT-R:5`-aatgaaacactttcaggt-3`, and test needed primer 18S-qRT-F:5`-tcttcttagagggattagcgg-3` and 18S-qRT-R:5`-aaggggattgaacgggtta-3` as the real-time fluorescence quantitative PCR of the Procambius clarkii 18S rRNA gene of internal reference, the SYBR Premix EX Taq(Perfect Real time dye method real time fluorescent quantitative test kit that utilizes precious biotechnology (Dalian) company limited to provide) carry out the real-time fluorescence quantitative PCR reaction, wherein, the real-time thermal cycler real-time fluorescence quantitative PCR instrument that the PCR instrument provides for U.S. Bio-Rad company, the reaction cumulative volume is 10 μ L, concrete application of sample amount is carried out according to the specification sheets of SYBR Premix EX Taq, preferred reaction conditions is: at first will be at 95 ℃ of denaturation 3min, be the cycle stage of repeatedly carrying out afterwards, each circulation is set to: 95 ℃ of sex change 30s, 60 ℃ of annealing 35s, and carrying out fluorescence numerical value from 55 ℃-95 ℃ progresses according to 0.5 ℃ of per second rising reads, obtain the Ct value that each sampling time point is corresponding,
7) data analysis: the Ct value corresponding to each sampling time point in step (6) carried out statistical study, the hsp70 gene of Procambius clarkii and 18S rRNA gene all can obtain corresponding Ct value, 3 groups of parallel tests will obtain respectively 3 groups of Ct values, finally need to calculate mean value separately, afterwards, using the average Ct value of Procambius clarkii 18S rRNA gene as reference, and the hsp70 gene average Ct value corresponding to all time points carried out data processing, and concrete grammar is: the form of calculation of data is 2
-Δ Ct, the average Ct value of the Δ Ct=(hsp70 gene of each time point-average Ct value of 18S rRNA gene wherein), utilize afterwards the excell data analysis tool, using the time of coercing as X-coordinate, with 2
-Δ Ctvalue is drawn column diagram as ordinate zou, simultaneously, adopts the significant difference between the data analysing method analytical data of T check, with normal Procambius clarkii of through sewage, coercing stimulation as a control group, is the 0h control group of coercing stimulation, with 2 of 0h
-Δ Ctvalue in contrast, sampling time point 6h, 12h, 24h, 48h, 72h, 96h corresponding 2
-Δ Ctvalue compares with it, analyze the rise situation of hsp70 gene relative expression quantity, and, preferred T check data analytical significant difference, when the highest up-regulated expression amount of hsp70 gene is compared (the P value in variance analysis<0.05 is designated as *) while having significant difference with the 0h control group, define so Wastewater Sample and have comparatively serious ecological toxicity harm, be pollution comparatively serious, when the highest up-regulated expression amount of hsp70 gene is compared (the P value in variance analysis<0.01 while having utmost point significant difference with the 0h control group, be designated as * *), define so Wastewater Sample and have very serious ecological toxicity harm, be pollution very serious.
Advantage of the present invention:
The present invention adopts the degree of the method qualitative evaluation water pollution of gene expression pattern, can analyse in depth the potential ecological toxicological effect existed after water pollution, and the present invention is practical, operate comparatively easyly, is suitable for the use of environment monitoring department.
The accompanying drawing explanation
Fig. 1 is that sewage is coerced the expression pattern figure of the rear Procambius clarkii hsp70 gene of stimulation with respect to 18S rRNA gene.
Embodiment
Embodiment 1:
Utilize biological method to carry out the Water quality evaluation that the Tailings Dam leachate is selected in certain metal smelting
In Fig. 1, Y value is 2
-Δ Ct, X-coordinate numerical value is the time, * means that this numerical value compares with Normal group, and significant difference, P value<0.005, * * means that this numerical value compares with Normal group, difference is extremely remarkable, P value<0.001.
The Water quality evaluation method is as follows:
1) precuring of Procambius clarkii: from fish market, buy 90 of Procambius clarkiis (20g-30g) alive, be divided into 3 groups, raise 1 day-2 days with the tap water of appropriate process sterilising treatment in aquarium, allow its adequacy test environment, obtain the precuring Procambius clarkii;
2) pre-treatment of detected Wastewater Sample: will gather certain the metal smelting come and select Tailings Dam leachate water sample to carry out 5 times of dilutions with distilled water, and be equally divided into 3 parts, and obtain 3 groups of water samples after diluting;
3) Procambius clarkii coerce irritant test: the precuring in step (1) is coerced to stimulation in 3 parts of water samples after dilution in step (2) for Procambius clarkii, and beginning timing, every group stays 3 less than the normal Procambius clarkii through stimulation oversaturation as a control group in advance simultaneously, sampling time point is designated as 0h, according to getting 3 of shrimps alive the sample time of 6h, 12h, 24h, 48h, 72h, 96h from every group, obtain the Procambius clarkii of coercing stimulation that each sampling time point is corresponding afterwards;
4) extraction of the total RNA of Procambius clarkii: the Procambius clarkii of coercing stimulation corresponding to each sampling time point of collecting in step (3) carried out to the liver sampling, the sampling amount that requires every Procambius clarkii liver is 20mg-30mg, and after the liver organization that requires to get on a sampling time point 3 Procambius clarkiis mixes, the Total RNA Extractor(Trizol that utilizes Shanghai living work biotechnology company limited to provide) the quick extraction agent box of the total RNA of animal carries out the extraction of total RNA, concrete grammar is: the homogenizer that the liver compound sample of every group of time point is placed in to 5mL, the Trizol extracting solution that respectively adds 1mL, carry out homogenized, nucleoprotein is separated fully with nucleic acid, after homogenized, homogenate is poured in the clean centrifuge tube of 1.5mL, and add the 0.2mL chloroform in centrifuge tube, thermal agitation 15s, room temperature is placed 3min, then, under 10000rpm-12000rpm and 4 ℃ of conditions, centrifugal 5min-10min, draw the upper water phase transition to clean centrifuge tube, add respectively isopyknic Virahol, mix, place 20min under room temperature condition, then under 10000rpm-12000rpm and 4 ℃ of conditions, centrifugal 5min-10min, abandon supernatant, in each centrifuge tube, add again 75% ethanol of 1mL to carry out washing precipitation, under 10000rpm-12000rpm and 4 ℃ of conditions, the quick centrifugal 3min-5min of low temperature, after discarding supernatant, seasoning 5min-10min under room temperature condition, finally, the RNase-free ddH that adds respectively 20 μ L
2o, fully dissolve RNA and be the total RNA sample of Procambius clarkii that each sampling time point is corresponding,
5) reverse transcription of Procambius clarkii cDNA is synthetic: by each time point in step (4), the corresponding total RNA sample of Procambius clarkii utilizes Shanghai to give birth to the single stage method RT-PCR amplification kit (M-MuLV) that work biotechnology company limited provides, the reverse transcription of carrying out cDNA is synthetic, and treatment process is: in the Eppendorf tube of nuclease free, add Oligo (dT)
20(50 μ M) 1 μ L, total RNA sample liquid 2 μ L, 10mM dNTP mixture 1 μ L, RNase-free ddH
2o8 μ L, and, after this mixture is placed in to 65 ℃ of water-bath heating 5min-10min, be placed in rapidly cooling 5min-10min on ice bath, after of short duration low-speed centrifugal, add 5 * the first chains to synthesize damping fluid 4 μ L, 0.1M DTT2 μ L, RNaseOUT
tM nucleic acid inhibitor 1 μ L, and, in centrifuge tube, gently various compositions are mixed, hatch 2min-3min in 37 ℃ of water-baths, then add at ambient temperature the reversed transcriptive enzyme of 1 μ L, piping and druming mixes lightly, in 37 ℃ of water-baths, hatches 5min-10min, finally under 70 ℃ of water bath condition, heating 15min-20min is with termination reaction, and the sample of acquisition is the Procambius clarkii liver organization cDNA sample that each time point is corresponding;
6) Real-Time Fluorescent Quantitative PCR Technique is determined the expression pattern of Procambius clarkii hsp70 gene: Procambius clarkii liver organization cDNA sample corresponding to each time point of usining in step (5) is as template, the real-time thermal cycler real-time fluorescence quantitative PCR instrument that utilizes U.S. Bio-Rad company to provide carries out the real-time fluorescence quantitative PCR test, the SYBR Premix EX Taq(Perfect Real time dye method real time fluorescent quantitative test kit that reaction adopts precious biotechnology (Dalian) company limited to provide) carry out, cumulative volume is 10 μ L, the application of sample amount is carried out with reference to SYBR Premix EX Taq specification sheets, two pairs of primers that use are respectively hsp70-qRT-F:5`-caccagatgaaggagttg-3` and hsp70-qRT-R:5`-aatgaaacactttcaggt-3`, and 18S-qRT-F:5`-tcttcttagagggattagcgg-3` and 18S-qRT-R:5`-aaggggattgaacgggtta-3`, specific procedure is: 95 ℃ of denaturation 3min, 40 circulations: 95 ℃ of sex change 30s, 60 ℃ of annealing 35s, and carrying out fluorescence numerical value from 55 ℃-95 ℃ progresses according to 0.5 ℃ of per second rising reads, obtain solubility curve corresponding to reaction process, reading of data can obtain the Ct value that each sampling time point is corresponding,
7) data analysis: Ct value corresponding to each sampling time point obtained in step (6) adopted to 2
-Δ Ctform means, utilizes the data analysis method of T check to carry out the significance analysis employing, wherein, with normal Procambius clarkii of through sewage, coercing stimulation as a control group, is the 0h that coerces stimulation, and concrete grammar is: with 2 of 0h
-Δ Ctvalue in contrast, sampling time point 6h, 12h, 24h, 48h, 72h, 96h corresponding 2
-Δ Ctvalue compares with it, analyze in each sampling time point the hsp70 gene rise situation of high relative expression quantity, and, utilize the data analysing method of T check to analyze significant difference, when the highest up-regulated expression amount of hsp70 gene is compared with the 0h control group while having significant difference, be that P value in variance analysis<0.05 is designated as *, define so Wastewater Sample and have comparatively serious ecological toxicity harm, be that water pollution is comparatively serious, when the highest up-regulated expression amount of hsp70 gene is compared with the 0h control group while having utmost point significant difference, it is P value in variance analysis<0.01, be designated as * *, define so Wastewater Sample and have very serious ecological toxicity harm, pollute very serious, result shows: in this experiment each sampling time point corresponding 2
-Δ Ctnumerical value and see the following form 1 with respect to the changing conditions of Normal group (0h control group), shown respectively 2 of sample time, freshwater crayfish MT gene relative expression quantity that each sampling time point is corresponding
-Δ Ctnumerical value, and with respect to 2 of Normal group (0h control group) freshwater crayfish
-Δ Ctthe changing conditions of numerical value, wherein, after sewage is coerced and is stimulated 24h, with the 0h Normal group, compare, the relative expression quantity of hsp70 gene raises 4.664 times, and there is significant difference between data, after sewage is coerced and is stimulated 48h, with Procambius clarkii 0h Normal group, compare, the highest relative expression quantity of hsp70 gene raises 7.822 times, and there is utmost point significant difference between data, therefore, significant difference implementations according to the highest up-regulated expression amount of hsp70 gene, we assert that examined water sample pollution condition is very serious, there is very serious ecological toxicity harm, should administer at once.
Each sampling time point of table 1 corresponding 2
-Δ Ctnumerical value and changing conditions
Claims (4)
1. the method for a biological assessment polluted water weight, is characterized in that, adopts the expression pattern of hsp70 gene to carry out the Water quality evaluation.
2. the method for a kind of biological assessment polluted water weight according to claim 1, is characterized in that, comprises following steps:
1) precuring of Procambius clarkii: the Procambius clarkii of the work that to get body weight be 20g-30g, in testing laboratory's aquarium, with the tap water through sterilising treatment, raise 1 day-2 days, obtain the precuring Procambius clarkii;
2) pre-treatment of Wastewater Sample: will gather the Wastewater Sample come and be diluted, and be equally divided into 3 groups, and obtain 3 groups of water samples after diluting;
3) Procambius clarkii coerce irritant test: the precuring Procambius clarkii in step (1) is put among 3 groups of water samples after step (2) dilution, coerced stimulation, sampling time point is respectively coerces 6h, 12h, 24h, 48h, 72h, the 96h stimulated after starting, and obtains the Procambius clarkii of coercing stimulation that each sampling time point is corresponding;
4) extraction of the total RNA of Procambius clarkii: the Procambius clarkii of coercing stimulation corresponding for each sampling time point in step (3) carries out the liver sampling, the sampling amount that requires every Procambius clarkii liver is 20mg-30mg, and after the liver organization that requires to get on a sampling time point 3-5 Procambius clarkii mixes, carry out the extraction of total RNA, obtain the Procambius clarkii liver total RNA sample of coercing stimulation that each sampling time point is corresponding;
5) reverse transcription of Procambius clarkii cDNA is synthetic: by each sampling time point in step (4), the corresponding Procambius clarkii liver total RNA sample of coercing stimulation carries out the synthetic cDNA of reverse transcription, obtains the Procambius clarkii liver organization cDNA sample that each sampling time point is corresponding;
6) Real-Time Fluorescent Quantitative PCR Technique is determined the expression pattern of Procambius clarkii hsp70 gene: Procambius clarkii liver organization cDNA sample corresponding to each sampling time point of usining in step (5) is as template, carry out the real-time fluorescence quantitative PCR reaction, the primer used is respectively the pair of primers hsp70-qRT-F:5`-caccagatgaaggagttg-3` of hsp70 gene and hsp70-qRT-R:5`-aatgaaacactttcaggt-3` and as pair of primers 18S-qRT-F:5`-tcttcttagagggattagcgg-3` and the 18S-qRT-R:5`-aaggggattgaacgggtta-3 of the 18S rRNA gene of internal reference, after the real-time fluorescence quantitative PCR reaction finishes, obtain solubility curve corresponding to reaction process, and carry out Ct value reading, obtain the Ct value that each sampling time point is corresponding,
7) data analysis: Ct Value Data corresponding to each sampling time point obtained in step (6) analyzed, usingd normal Procambius clarkii of through sewage, not coercing stimulation as the 0h control group, specific analytical method is: the form of calculation of data is 2
-Δ Ct, the average Ct value of the Δ Ct=(MT gene of each sampling time point-average Ct value of 18S rRNA gene wherein), afterwards with 2 of 0h control group
-Δ Ctvalue in contrast, sampling time point 6h, 12h, 24h, 48h, 72h, 96h corresponding 2
-Δ Ctvalue compares with it, analyze the rise situation of hsp70 gene relative expression quantity, when the highest up-regulated expression amount of hsp70 gene is compared with the 0h control group while having significant difference, be that P value in variance analysis<0.05 is designated as *, define so Wastewater Sample and have comparatively serious ecological toxicity harm, be that water pollution is comparatively serious, when the highest up-regulated expression amount of hsp70 gene is compared with the 0h control group while having utmost point significant difference, it is P value in variance analysis<0.01, be designated as * *, define so Wastewater Sample and have very serious ecological toxicity harm, pollute very serious.
3. the method for a kind of biological assessment polluted water weight according to claim 1, it is characterized in that, real-time fluorescence quantitative PCR adopts the reaction conditions of concrete amplification program to be: 95 ℃ of denaturation 3min, be the cycle stage of repeatedly carrying out afterwards, each circulation is set to: 95 ℃ of sex change 30s, 60 ℃ of annealing 35s, and carry out fluorescence numerical value from 55 ℃-95 ℃ progresses according to 0.5 ℃ of per second rising and read, solubility curve corresponding to reaction process obtained.
4. the method for a kind of biological assessment polluted water weight according to claim 1, is characterized in that, utilize data analysing method analytical sampling time point 6h, 12h, 24h, 48h, 72h, the 96h of T check corresponding 2
-Δ Ctbe worth corresponding with the 0h control group 2
-Δ Ctthe p value of significant difference between value, come the expression pattern of qualitative evaluation Procambius clarkii hsp70 gene and the relation between the water sample pollution level.
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| CN108883417A (en) * | 2016-01-20 | 2018-11-23 | 特里夫科技公司 | Instant nucleic acid amplification and detection |
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Application publication date: 20131218 |