CN102181518A - Method for identifying marine water fecal pollution source by using genetic finger-print of Escherichia coli - Google Patents
Method for identifying marine water fecal pollution source by using genetic finger-print of Escherichia coli Download PDFInfo
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Abstract
The invention provides a method for identifying marine water fecal pollution source by using a genetic finger-print of Escherichia coli, which comprises the following steps of: separating and purifying Escherichia coli, establishing a genetic finger-print library of the Escherichia coli from a known source and analyzing a marine water fecal pollution source. In the method, the Escherichia coli is separated and purified by a filter membrane method, the genetic finger-print library of the Escherichia coli from a known fecal source is established by selecting a repetitive extragenic palindromic sequence polymerase chain reaction, and a genetic finger-print, which is separated from a marine water sample, of Escherichia coli from an unknown source is compared with a genetic finger-print library of Escherichia coli from the known fecal source, so that the most possible source of the Escherichia coli is determined according to the sequence of similarity to indentify the fecal pollution source in marine water. The method is simple in required instruments and equipment, short in detection time and high in resolvability, repeatability and stability, can be used for detecting a large batch of samples and can meet the requirements on the monitoring and management of fecal pollution of off-shore sea areas.
Description
Technical field
The present invention relates to a kind of method of utilizing bacillus coli gene finger printing identification ocean water body fecal pollution source, specifically, relate to the trace to the source method of utilizing bacillus coli gene finger printing identification ocean water body fecal pollution source of analytical procedure of a kind of establishment step that comprises intestinal bacteria purification procedures, bacillus coli gene finger printing storehouse, known source and ocean water body fecal pollution.
Background technology
(Microbial source tracking MST) at first rises and progressively development in countries such as America and Europes the eighties in 20th century microbial source tracer method.MST utilizes biochemical characteristic, genetic diversity and the specific metabolic product thereof of microorganism in environment to determine a kind of technology in host bacterium source, and it has especially played incomparable effect to the monitoring of non-point source fecal pollution with management.The ultimate principle of MST is: live in same a kind of microorganism in the different host environments in order to adapt to residing specificity ecotope, a series of environmental compatibilities have been produced, and with these characteristics to pass to the descendants, and then form a micropopulation of the same race that is different from other environment, these microorganisms can form and adapt to living environment specificity finger printing, by analyzing the purpose that these specificity finger printings just can reach identification microbial contamination source.The basic line of MST compares with the finger printing of sample and the specificity collection of illustrative plates in the java standard library at first setting up known substance provenance indicator organism specificity finger printing storehouse, with the ight soil source of the detailed identification of homology likelihood polluted-water.
The spike factor commonly used---the intestinal bacteria of MST.Intestinal bacteria (E.coli) are colon bacillus, and (Escherichia) represents bacterium for Escherichia.Intestinal bacteria behaviour waits and often to occupy bacterium in the homoiothermy animal intestinal, and its be everlasting baby or nascent animal birth back several hrs can enter digestive tube from the oral cavity, and a large amount of breedings and existing throughout one's life in gastral rear end.Intestinal bacteria are owing to often excrete with ight soil, and the energy wide-scale distribution is in external environment, so intestinal bacteria are one of important indicators of water quality monitoring (WQM) in the world.The intestinal bacteria molecular structure is comparatively simple, and its DNA is a complete DNA chain, and as molecular biological pattern bacterium, the mankind study comparatively thoroughly to it.Therefore, comprehensive above factor, intestinal bacteria are elected as the first-selected spike factor of MST usually by people.
MST fingerprint pattern technology--REP-PCR TRAP.Gene fingerprint is the flag sequence with pcr amplification environmental microorganism sample total DNA, the collection of illustrative plates with particular bands feature that its separation is formed with suitable electrophoretic technique then.The difference of genetic fingerprint pattern just reflects the difference of bacterium kind.Bacterial genomes tumor-necrosis factor glycoproteins pcr amplification method (PCR of repetitive sequences, rep-PCR) be a kind of bacterial genomes fingerprinting method that Versalovic described in 1996, it utilizes the dna primer complementation to amplify that nature is that exist, high conservative, (these sequences are the multiple copied sequence to the repeated fragment dna sequence dna, in Gram-negative bacteria, exist mostly, and in gram-positive microorganism, seldom exist), by the electrophoretic band comparative analysis, disclose the difference between genome.(repetitive extragenic palindromic REP) is a kind of main type of tumor-necrosis factor glycoproteins to the outer palindromic sequence of the repeated gene of 35-40bp.This technology of REP-PCR is with a high credibility, favorable reproducibility, fast, resolving power is high, is the genotype fingerprint pattern technology of relatively praising highly in the MST research always.
Summary of the invention
The object of the invention provides a kind of method of utilizing intestinal bacteria rep-PCR gene fingerprint identification ocean water body fecal pollution source, and this method comprises:
The intestinal bacteria purification procedures, in this step, select the fecal sample of suitable gradient volume, adopt filter membrane method, with band raster fibrous filter membrane and m-TEC nutrient agar is after matrix is carried out the strain separating cultivation, picking meets the bacterium colony of intestinal bacteria morphological specificity, is that matrix is carried out purifying 2～3 times with the LB nutrient agar, preserves be defined as colibacillary bacterial strain after biochemical identification;
The establishment step in bacillus coli gene finger printing storehouse, known source, in this step, adopt traditional phenol-chloroform extraction method to extract bacillus coli gene group DNA, carry out polymerase chain reaction with specific primer, adopt damping fluid, staining fluid and sepharose to carry out the polymerase chain reaction product electrophoresis, adopt the irradiation of camera bellows ultraviolet transmissive to carry out the sepharose imaging, thereby obtain the gene fingerprint of each bacterial strain, make up bacillus coli gene finger printing storehouse, known source;
The ocean water body fecal pollution analytical procedure of tracing to the source, in this step, adopt data analysis software, to separate the unknown source bacillus coli gene finger printing and the bacillus coli gene finger printing storehouse, known source that obtain from the ocean water body sample compares, determine its maximum possible source according to the ordering of similarity size, and in view of the above the fecal pollution in the water body of sampling marine site is traced to the source.
Intestinal bacteria purification procedures of the present invention comprises:
1. select the fecal sample of suitable gradient volume, adopt filter membrane method, with the m-TEC nutrient agar is after matrix is carried out the strain separating cultivation, picking meets the bacterium colony of intestinal bacteria morphological specificity, with the LB nutrient agar is that matrix is carried out purifying 2～3 times, preserves be defined as colibacillary bacterial strain after biochemical identification;
2. in the intestinal bacteria biochemical identification, all need carry out oxidase test, the test of western Meng Shi citrate, indole test, the fermentation test of EC meat soup totally 4 kinds of experiments to every strain bacterium, what 4 kinds of requirement of experiment were all satisfied fully is intestinal bacteria.
Intestinal bacteria purification procedures of the present invention can the mass disposal fecal sample, realizes colibacillary sharp separation and purifying.
The establishment step in intestinal bacteria rep-PCR gene fingerprint storehouse, known source of the present invention comprises:
1. bacillus coli gene group DNA extraction method adopts traditional phenol-chloroform extraction method (needing the RNA lytic enzyme) and commercially available reagent box method;
2.PCR the PCR primer sequence that amplified reaction is selected for use is
REP?1R：5’-IIIICGICGICATCIGGC-3’；
REP 2I:5 '-ICGICTTATCIGGCCTAC-3 ' (I is an xanthoglobulin).
3.PCR the product electrophoresis adopts 0.5 * tbe buffer liquid (or 0.5 * TAE damping fluid); The DNA staining fluid adopts ten thousand/ nucleic acid dye, Gene Finder for example, or suitable other DNA staining fluid of level; Sepharose requires length 12cm, the comb facewidth 〉=4mm; Applied sample amount is 10 μ L; Voltage stabilized source is carried out electrophoresis.
4. the irradiation of camera bellows ultraviolet transmissive is adopted in the sepharose imaging, and pixel is more than 1,000,000 grades.
5. spectrum library analytical procedure application data analysis software adopts techniques of discriminant analysis that spectrum library is analyzed, and assesses the classification accuracy in intestinal bacteria rep-PCR gene fingerprint storehouse, known source with the average correct classification rate that the source is verified and cross validation obtains.
The foundation and the analytical procedure in intestinal bacteria rep-PCR gene fingerprint storehouse, known source of the present invention can be used for the colibacillary rep-PCR genetic fingerprint of mass detection, can satisfy spectrum library is set up and the ageing and accuracy requirement of analyzing, the quick foundation that can be used for spectrum library is analyzed with accurate.
The ocean water body fecal pollution of the present invention analytical procedure of tracing to the source comprises:
In the method, to from the ocean water body sample, separate the colibacillary rep-PCR gene fingerprint of the unknown source that obtains and known source intestinal bacteria rep-PCR gene fingerprint is compared, the discriminant analysis program of application data analysis software is carried out the classification identification of unknown source bacterial strain, determine its maximum possible source according to the ordering of similarity size, and in view of the above the fecal pollution in the water body of sampling marine site is traced to the source.
Ocean water body fecal pollution of the present invention is traced to the source, and analytical procedure can be used for accurately, efficient identification ocean water body fecal pollution source, provides foundation for the monitoring of immediate offshore area fecal pollution with management timely and effectively.
The advantage applies of the inventive method exists:
The accuracy height in identification ocean water body fecal pollution source; Plant and instrument required for the present invention is simple, and detection time is short; Can carry out pattern detection in enormous quantities; Distinguishing is good, repeatability is strong, stability is high.
Embodiment
The intestinal bacteria purification procedures that the present invention preferably adopts comprises:
(1) fecal sample collection: take the fresh excreta sample to put into aseptic test tube with ground stopper with aseptic sampling spoon, preserve transportation for 4 ℃, and in 24 hours, handle.
(2) sample pre-treatment: add stroke-physiological saline solution in 1: 10 ratio, Glass rod grinding, 5 minutes mixings of vortex vibrator vibration are diluted to suitable gradient with stroke-physiological saline solution;
(3) filter membrane suction filtration: carry out suction filtration with 0.45 μ m band raster fibrous filter membrane, the filter membrane that suction filtration is good is attached on the m-TEC nutrient agar flat board, cultivates 2h under 35 ± 0.5 ℃ of conditions, is put in 44.5 ± 0.5 ℃ the water-bath again and cultivates 22～24h;
(4) bacterial strain purifying: with the LB nutrient agar is matrix, and the little prominent single bacterium colony line of picking yellow, yellow-green colour or tawny and central authorities separates, and carries out 2～3 times purifying.
(5) biochemical identification: intestinal bacteria behind the purifying are carried out oxidase test, the test of western Meng Shi citrate, indole test, the fermentation test of the EC meat soup biochemical identification of totally 4 kinds of experiments.
(6) bacterial strain is preserved: the bacterial strain that will meet 4 kinds of biochemical identification experimental index fully is configured to contain the bacterium liquid of 30% glycerine ,-20 ℃ of freezing preservations.
The establishment step in intestinal bacteria rep-PCR gene fingerprint storehouse, known source of the present invention comprises:
(1) colibacillary purifying: it is streak culture to adopt nutrient agar medium that the intestinal bacteria bacterial classification is carried out, and culture temperature is 36 ± 1 ℃, and incubation time is 18～24h, sets up the negative contrast of blank nutrient agar medium plate; With choosing acicula picking list bacterium colony to LB liquid nutrient medium shaking culture, culture temperature is 36 ± 1 ℃, and incubation time is 16～18h, and the vibration rotating speed is 200rpm/min, sets up the negative contrast of blank LB nutrient solution; Get 10 μ L cultures and carry out LB substratum secondary shaking culture, culture temperature is 36 ± 1 ℃, and incubation time is 16h, and the vibration rotating speed is 200rpm/min, sets up the negative contrast of blank LB nutrient solution.
(2) extraction of bacillus coli gene group DNA: can adopt traditional phenol-chloroform extraction method and commercially available reagent box method; Get 4mL intestinal bacteria LB secondary culture and carry out the extraction of genomic dna, the assorted band of clear, the single nothing of bacillus coli gene group DNA band in 1% agarose electrophoresis, the molecular weight size that require to be extracted reach more than the 105bp, concentration reaches more than the 200ng/ μ L, (OD260-OD320)/(OD280-OD320) is 1.8-2.0.Phenol-the chloroform extraction method step is as follows for tradition: get 2mL intestinal bacteria LB secondary culture, the centrifugal 2min of 12000rpm/min in whizzer abandons supernatant and stays bacterium cake (supernatant will be removed totally as far as possible); Add 2mL intestinal bacteria LB secondary culture again, the same operation; Add 500 μ L dH2O, vibration 5min fully suspends it; Add 50 μ L Proteinase Ks (concentration of Proteinase K is 20mg/mL) and 100 μ L SDS (concentration of SDS is 10%) in suspension, mend dH2O to 1000 μ L, the mixing that turns upside down is placed on 1h in 65 ℃ of water-baths (or spending the night in 37 ℃ of water-baths); Add isopyknic PCI reagent (PCI is the saturated phenol of Tris: chloroform: the primary isoamyl alcohol volume ratio is 25: 24: 1), mixing 15min turns upside down, place the centrifugal 15min of whizzer 12000rpm/min, draw supernatant liquid (being sure not to bring into the liquid of middle level and lower floor), repeat the PCI operation; Draw supernatant liquid, add isopyknic CI reagent (CI is a chloroform: the primary isoamyl alcohol volume ratio is 24: 1), the mixing 15min that turns upside down places the centrifugal 15min of whizzer 12000rpm/min, draws supernatant liquid (being sure not to bring into the liquid of middle level and lower floor); Add 100% ice ethanol of NaAc (NaAc concentration is 3mol/L, pH value 5.2) and 2.5 times of volumes of 1/10 volume, the mixing that turns upside down places-80 ℃ of refrigerator 10min (or-20 ℃ of refrigerator 30min); Place 4 ℃ of centrifugal 15min of whizzer 12000rpm/min, slow sucking-off liquid keeps precipitation (action must be soft, be sure not to take away precipitation, precipitates sometimes naked eyes and cannot see); Add 70% ice ethanol, the mixing that turns upside down places 4 ℃ of centrifugal 5min of whizzer 12000rpm/min, and slow sucking-off liquid keeps precipitation (action must be soft, be sure not to take away precipitation, precipitates sometimes naked eyes and cannot see); Natural air drying 1～2h (or placing the dry 3min of Rotary drying instrument); Add 100 μ L TE damping fluids, fully mixing; It is standby to place 4 ℃ of refrigerators (or-20 ℃ of refrigerators) to preserve.
(3) pcr amplification reaction: the PCR primer sequence is
REP?1R：5’-IIIICGICGICATCIGGC-3’；
REP 2I:5 '-ICGICTTATCIGGCCTAC-3 ' (I is an xanthoglobulin).
The PCR reaction solution consists of primer REP 1R (100 μ M) 1 μ L, primer REP2I (100 μ M) 1 μ L, dNTP mixture (each 2.5mM) 8 μ L, LA Taq (5U/ μ L) 0.5 μ L, 10 * LA PCR damping fluid (Mg2+25mM), 5 μ L, template (bacillus coli gene group DNA) 50ng, dH
2O complements to 50 μ L.Reaction conditions is: 95 ℃ of pre-sex change 7min; 30 circulations that circulate then, each circulation is 94 ℃ of sex change 30sec, 49.4 ℃ of annealing 1min, 72 ℃ are extended 8min; Last 72 ℃ are extended 16min.The positive PCR that sets up the reference culture genomic dna is in the same old way and the negative control sample supplied with dH2O of template.
(4) PCR product electrophoresis: adopt 0.5 * tbe buffer liquid (or 0.5 * TAE damping fluid); 1.5% concentration sepharose (adding ten thousand/Gene Finder staining fluid doubly or the above DNA staining fluid of same level), sepharose requires length 12cm, the comb facewidth 〉=4mm; Applied sample amount is 10 μ L; Electrophoresis is after 15～20 minutes under the voltage of voltage regulation 280V, again with voltage of voltage regulation 200V electrophoresis 1～1.5h, to the swimming of 100bp Marker band to the gel bottom till.
(5) sepharose imaging: place gel imaging system to carry out imaging the sepharose behind the electrophoresis.Imaging system requires to be camera bellows ultraviolet transmissive irradiation (or blue light illumination) that pixel reaches more than 1,000,000 grades.
(6) finger printing is built the storehouse: will test the intestinal bacteria rep-PCR gene fingerprint input microbial source spike MST analytical system that obtains, and, set up intestinal bacteria rep-PCR gene fingerprint storehouse, known source with the processing of marking of 100bpDNA ladderMarker.
(7) spectrum library analysis: using microbe source spike MST analytical system is carried out discriminatory analysis to spectrum library, assesses the classification accuracy in intestinal bacteria rep-PCR gene fingerprint storehouse, known source with the average correct classification rate that the source is verified and cross validation obtains.
The ocean water body fecal pollution of the present invention analytical procedure of tracing to the source comprises:
Discriminant analysis program in the using microbe source spike MST analytical system, to from the ocean water body sample, separate the colibacillary rep-PCR gene fingerprint of the unknown source that obtains and compare in known intestinal bacteria rep-PCR gene fingerprint storehouse, determine its maximum possible source according to the ordering of similarity size, and in view of the above the fecal pollution in the water body of sampling marine site is traced to the source.
Selected reagent and material preferably are respectively in the inventive method:
Strains separation matrix: m-TEC nutrient agar
Bacterial strain purifying matrix: LB nutrient agar
Enrichment liquid: LB meat soup
Filter membrane: 0.45 μ m band raster fibrous filter membrane
Diluent: 0.9% physiological saline
PCR primer sequence: REP 1R:5 '-IIIICGICGICATCIGGC-3 '; REP 2I:5 '-ICGICTTATCIGGCCTAC-3 ' (I is an xanthoglobulin)
The PCR reaction solution: the PCR reaction solution consists of primer REP 1R (100 μ M) 1 μ L, primer REP 2I (100 μ M) 1 μ L, dNTP mixture (each 2.5mM) 8 μ L, LATaq (5U/ μ L) 0.5 μ L, 10 * LA PCR damping fluid (Mg2+25mM), 5 μ L, template (bacillus coli gene group DNA) 50ng, dH
2O is supplemented to 50 μ L.
Damping fluid: 0.5 * tbe buffer liquid
The DNA staining fluid: ten thousand/ Gene Finder
Mode by the following examples specifies the method that the present invention utilizes intestinal bacteria rep-PCR gene fingerprint identification ocean water body fecal pollution source.
Strains separation purifying embodiment (colibacillary separation and purification)
Colibacillary separation and purification: gather the fresh excreta sample, behind grinding and gradient dilution, carry out suction filtration with 0.45 μ m band raster fibrous filter membrane, the filter membrane that suction filtration is good is attached on the m-TEC nutrient agar flat board, cultivate 2h under 35 ± 0.5 ℃ of conditions, be put into again in 44.5 ± 0.5 ℃ the water-bath and cultivate 22～24h; The little prominent single bacterium colony of picking yellow, yellow-green colour or tawny and central authorities is that matrix is carried out 2～3 times purifying with the LB nutrient agar; Preserve after biochemical identification, being defined as colibacillary bacterial strain, finally obtain the coli strain of 573 strains, comprise 159 strains of people source, 108 strains of dog source, 95 strains of chicken source, 112 strains of ox source, 99 strains of pig source from five kinds of different plant species sources.
Build storehouse embodiment (foundation of intestinal bacteria rep-PCR gene fingerprint storehouse, known source)
The foundation in intestinal bacteria rep-PCR gene fingerprint storehouse, known source: adopt traditional phenol-chloroform extraction method to extract bacillus coli gene group DNA, carry out pcr amplification reaction with the PCR primer, adopt damping fluid, Gene FinderDNA staining fluid and sepharose to carry out PCR product electrophoresis, adopt the irradiation of camera bellows ultraviolet transmissive to carry out 2,200,000 pixel sepharose imagings.With the 573 strain intestinal bacteria rep-PCR gene fingerprints input microbial source spike MST analytical system that obtains, and, set up intestinal bacteria rep-PCR gene fingerprint storehouse, known source with the processing of marking of 100bpDNA ladderMarker; Select for use discriminant analysis program that spectrum library is sorted out the accuracy checking.Poor＜5% of the source checking of this spectrum library species source module and the average correct classification rate of cross validation, it is good to illustrate that spectrum library is sorted out accuracy, puts into practice available.
The foundation of spectrum library of the present invention needs all left and right sides time, and each cycle can be carried out tens detections to up to a hundred samples, therefore can carry out the detection of sample in enormous quantities with the short time.
Claims (7)
1. method with bacillus coli gene finger printing identification ocean water body fecal pollution source, this method comprises:
The intestinal bacteria purification procedures, in this step, select the fecal sample of suitable gradient volume, adopt filter membrane method, with band raster fibrous filter membrane and m-TEC nutrient agar is after matrix is carried out the strain separating cultivation, picking meets the bacterium colony of intestinal bacteria morphological specificity, is that matrix is carried out purifying 2～3 times with the LB nutrient agar, preserves be defined as colibacillary bacterial strain after biochemical identification;
The establishment step in bacillus coli gene finger printing storehouse, known source, in this step, adopt traditional phenol-chloroform extraction method to extract bacillus coli gene group DNA, carry out polymerase chain reaction with specific primer, adopt damping fluid, staining fluid and sepharose to carry out the polymerase chain reaction product electrophoresis, adopt the irradiation of camera bellows ultraviolet transmissive to carry out the sepharose imaging, thereby obtain the gene fingerprint of each bacterial strain, make up bacillus coli gene finger printing storehouse, known source;
The ocean water body fecal pollution analytical procedure of tracing to the source, in this step, adopt data analysis software, to separate the unknown source bacillus coli gene finger printing and the bacillus coli gene finger printing storehouse, known source that obtain from the ocean water body sample compares, determine its maximum possible source according to the ordering of similarity size, and in view of the above the fecal pollution in the water body of sampling marine site is traced to the source.
2. the method with bacillus coli gene finger printing identification ocean water body fecal pollution source according to claim 1, wherein in the intestinal bacteria biochemical identification of intestinal bacteria purification procedures, all need carry out oxidase test, the test of western Meng Shi citrate, indole test, the fermentation test of EC meat soup totally 4 kinds of experiments to every strain bacterium, what 4 kinds of requirement of experiment were all satisfied fully is intestinal bacteria.
3. the method with bacillus coli gene finger printing identification ocean water body fecal pollution source according to claim 1, wherein in the establishment step in bacillus coli gene finger printing storehouse, known source, the primer sequence that polymerase chain reaction is selected for use is
REP?1R：5’-IIIICGICGICATCIGGC-3’
REP?2I：5’-ICGICTTATCIGGCCTAC-3’
Wherein, I is an xanthoglobulin.
4. the method with bacillus coli gene finger printing identification ocean water body fecal pollution source according to claim 1, wherein in the establishment step in bacillus coli gene finger printing storehouse, known source, the polymerase chain reaction product electrophoresis adopts 0.5 * tbe buffer liquid or 0.5 * TAE damping fluid; The DNA staining fluid adopts ten thousand/ nucleic acid dye or suitable other DNA staining fluid of level; Sepharose requires length 12cm, the comb facewidth 〉=4mm; Applied sample amount is 10 μ L; Voltage stabilized source is carried out electrophoresis.
5. the method with bacillus coli gene finger printing identification ocean water body fecal pollution source according to claim 1, wherein in the establishment step in bacillus coli gene finger printing storehouse, known source, the irradiation of camera bellows ultraviolet transmissive is adopted in the sepharose imaging, and pixel is more than 1,000,000 grades.
6. the method with bacillus coli gene finger printing identification ocean water body fecal pollution source according to claim 1, wherein in the establishment step in bacillus coli gene finger printing storehouse, known source, to the analytical applications data analysis software of spectrum library, the average correct classification rate that adopts techniques of discriminant analysis to obtain with source checking and cross validation is assessed the classification accuracy in intestinal bacteria rep-PCR gene fingerprint storehouse, known source.
7. the method with bacillus coli gene finger printing identification ocean water body fecal pollution source according to claim 1 wherein in the ocean water body fecal pollution is traced to the source analytical procedure, adopts techniques of discriminant analysis to carry out the classification identification of unknown source bacterial strain.
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| CN104263806A (en) * | 2014-09-29 | 2015-01-07 | 青岛康合伟业商贸有限公司 | Fuchsin basic sodium sulfite agar and application thereof |
| CN104263676A (en) * | 2014-09-02 | 2015-01-07 | 青岛永通电梯工程有限公司 | Simmons citrate agar and application thereof |
| CN106399303A (en) * | 2016-09-23 | 2017-02-15 | 浙江省检验检疫科学技术研究院 | Host-enteric microbial interacting gene-based specific molecular marker 1-38 and detection method for pollution by excrement of pig |
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| CN104263676A (en) * | 2014-09-02 | 2015-01-07 | 青岛永通电梯工程有限公司 | Simmons citrate agar and application thereof |
| CN104263806A (en) * | 2014-09-29 | 2015-01-07 | 青岛康合伟业商贸有限公司 | Fuchsin basic sodium sulfite agar and application thereof |
| CN106399303A (en) * | 2016-09-23 | 2017-02-15 | 浙江省检验检疫科学技术研究院 | Host-enteric microbial interacting gene-based specific molecular marker 1-38 and detection method for pollution by excrement of pig |
| CN106399303B (en) * | 2016-09-23 | 2019-02-05 | 浙江省检验检疫科学技术研究院 | It is a kind of that specific molecular marker 1-38 and its detection method are polluted based on host-enteric microorganism interacting genes swine excrement |
| CN108531496A (en) * | 2018-04-04 | 2018-09-14 | 江南大学 | A kind of repeated palindromic sequence improving foreign gene mRNA and its application |
| CN108531496B (en) * | 2018-04-04 | 2020-11-06 | 江南大学 | DNA for increasing exogenous gene mRNA quantity and application thereof |
| CN113934875A (en) * | 2021-10-27 | 2022-01-14 | 云舟生物科技（广州）有限公司 | Electrophoresis data identification method and system, computer storage medium and electronic equipment |
| CN113934875B (en) * | 2021-10-27 | 2022-11-25 | 云舟生物科技(广州)股份有限公司 | Electrophoresis data identification method and system, computer storage medium and electronic equipment |
| 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 |
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