CN1563422A - Method for fast detecting colibacillus in drinking water - Google Patents
Method for fast detecting colibacillus in drinking water Download PDFInfo
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- CN1563422A CN1563422A CN 200410022322 CN200410022322A CN1563422A CN 1563422 A CN1563422 A CN 1563422A CN 200410022322 CN200410022322 CN 200410022322 CN 200410022322 A CN200410022322 A CN 200410022322A CN 1563422 A CN1563422 A CN 1563422A
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- 235000020188 drinking water Nutrition 0.000 title claims abstract description 6
- 239000003651 drinking water Substances 0.000 title claims abstract description 6
- 238000000034 method Methods 0.000 title abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 33
- 244000005700 microbiome Species 0.000 claims abstract description 11
- 238000012408 PCR amplification Methods 0.000 claims abstract description 7
- 238000000605 extraction Methods 0.000 claims abstract description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- 238000001962 electrophoresis Methods 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 10
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 9
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 229920000936 Agarose Polymers 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 7
- 229960005542 ethidium bromide Drugs 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 5
- 108010067770 Endopeptidase K Proteins 0.000 claims description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 101100275473 Caenorhabditis elegans ctc-3 gene Proteins 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 claims description 2
- 239000007984 Tris EDTA buffer Substances 0.000 claims 2
- 239000000872 buffer Substances 0.000 claims 1
- 239000012898 sample dilution Substances 0.000 claims 1
- 239000008223 sterile water Substances 0.000 claims 1
- 238000003752 polymerase chain reaction Methods 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract 1
- 238000010998 test method Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 17
- 239000008399 tap water Substances 0.000 description 11
- 235000020679 tap water Nutrition 0.000 description 11
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- 239000012530 fluid Substances 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 7
- 230000000968 intestinal effect Effects 0.000 description 7
- 238000002156 mixing Methods 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- 239000004141 Sodium laurylsulphate Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
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- 102000004190 Enzymes Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- -1 pcr amplification Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 208000034817 Waterborne disease Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
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- 230000001332 colony forming effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
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- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
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- 238000002347 injection Methods 0.000 description 1
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- 238000009434 installation Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
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- 238000010068 moulding (rubber) Methods 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 231100000004 severe toxicity Toxicity 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
This invention relates to a quick test method to drinking water colibacillus applying the polymerase chain reaction to test colibacillus with the PCR method including the following steps: collection of microorganism in water sample, extraction of microorganism gene group DNA, PCR amplification and survey to the product. Compared with the traditional method, this method uses only 4-5 hours to finish the test from sending the sample to the laboratory and can find out 1-10 colibacilluses/ML sample while the traditional method needs at least 48 hours.
Description
Technical field:
The invention belongs to drinking-water quality research, water-treatment technology field, be specifically related to the colibacillary molecular biology method for quick of a kind of tap water.
Background technology:
The microbiology index of tap water and people's is healthy closely bound up, is that drinking-water quality detects the index of very paying close attention to.The tap water microorganism detection method that generally adopts comprises direct census (microscopic count) method, live bacterial count (ColonyForming Units at present, CFU) method and most probable number (MPN) method etc., these methods have been brought into play enormous function to the bacteriology index of control tap water.But inevitably can there be certain defective in traditional detection method: (1) is (as medium component, culture condition etc.) for various reasons, and present educable microorganism is less than 1% of microbial count, makes detected result quite inaccurate.For example multitube fermentation method (MUG) is difficult to detect E.coli 0 157:H7; (2) detection time long, need 48h at least, can not satisfy at all modern society to waterborne disease find fast, the requirement of rapid detection.
Summary of the invention:
Tap water intestinal bacteria molecular Biological Detection method has been set up in polymerase chain reaction in the applied molecular biology of the present invention.Reaction primer of the present invention: upstream primer: 5 '-tgt tca gtg gca aga gtt-3 '
Downstream primer: 5 '-taa tcg a ta tac ccg ctc-3 '
Detect intestinal bacteria with PCR method, comprise following step: four steps such as the extraction of the collection of microorganism, microbe genome DNA in the water sample, pcr amplification, product observation.
1, the collection of microorganism reaches in the water sample
Adopt filter membrane or filter after sterilizing to filter water sample, in the EP pipe of sterilized water is housed, the washing filter membrane, centrifugal, abandon supernatant liquor, standby;
2, the extraction of microbe genome DNA in the sample:
A, in above-mentioned EP pipe, add the TE damping fluid, make it resuspended;
B, adding SDS and Proteinase K, mixing, incubation 45min～1h;
C, adding chloroform/primary isoamyl alcohol, mixing, centrifugal, supernatant liquor is changed in the new EP pipe;
D, add isopyknic phenol/chloroform/primary isoamyl alcohol, mixing, centrifugal, supernatant liquor is changed in the new EP pipe;
E, adding Virahol mix up to DNA precipitating gently, and be centrifugal;
F, abandon supernatant, the throw out washing with alcohol is dried;
G, will precipitate and heavily be dissolved in the TE damping fluid, shake up;
The OD value of H, genomic dna template that measure to extract with ultraviolet spectrophotometer is calculated its DNA concentration, and method of calculation are:
DNA concentration (μ g/ μ L)=OD value 260nm * diluted sample multiple * 50/1000
J, the genomic dna template is carried out mark, store, standby;
3, pcr amplification;
50 μ L reaction systems:
10 * PCR damping fluid, 5 μ L
Tag enzyme 2U/ μ L 1 μ L
MgCl2(25mM) 5μL
dNTP(2.5mM) 5μL
ddH20 29μL
Upstream primer 10 μ M 2 μ L
Upstream primer 10 μ M 2 μ L
Template DNA 1.5 μ g/ μ L 1 μ L
4, product is observed:
The preparation of A, gel: dispose agarose solution earlier, heating is fully dissolved it, adds ethidium bromide solution again;
B, configuration TAE electrophoresis liquid;
C, the glue that falls: the agarose solution that configures is poured in the offset plate, treat that it solidifies after, put into electrophoresis chamber;
D, electrophoresis: will increase good PCR product and damping fluid are mixed in proportion, and join in the gel well, add dna molecular amount standard simultaneously, connect power supply, setting voltage and electric current, beginning electrophoresis.
E, observation; Amplified production is observed pictures taken under UV-light.
Adopt above-mentioned tap water intestinal bacteria biomolecular science detection method to compare with traditional method, the time is short, delivers to the laboratory from water sample and begins to finish to detection, approximately needs 4～5 hours; Highly sensitive, can detect 1～10 intestinal bacteria/mL water sample.And traditional detection method needs 48h at least.
Description of drawings
Fig. 1 and Fig. 2 are the gel electrophoresis images of water sample pcr amplification product
Among the figure: 1 dna molecular amount standard; 2-15 is water sample microorganism PCR amplified production
Embodiment
Below be example with 15 water samples of Chongqing City's city planting ductwork, the specific implementation process of process in detail:
The first step: the extraction of microorganism in the water sample:
The Indicator for Drinking Water Quality regulation of 1 China, the standard of coliform is＜1/100mL in the tap water, so water sampling 100mL.
The sterilization of 2 filter membranes and filter and installation.Filter 121 ℃ of sterilizations of high pressure steam 20min.The edge of the 0.22 μ m filter membrane of having sterilized with aseptic tweezers gripping, uneven surface upwards paste and are placed on the sterilization filter bed.
3 with 100mL water sample injection filter, adds a cover, and opens the filter valve, suction filtration under negative 50KPa.
4 usefulness cotton ball soaked in alcohol wiping scissors with aseptic nipper gripping filter membrane edge, are cut into the slice of wide 0.5mm with filter membrane with scissors, put into the EP pipe that the 2.5mL sterilized water is housed, and shake the EP pipe gently, the washing filter membrane.
5 managed 15000r/m centrifugal 5 minutes with EP, abandoned supernatant, standby.
Second step: the genomic dna that extracts microorganism in the water sample:
The extracting method of microbe genome DNA:
1 medicine: EDTA, I mol/L Tris-Cl, TE damping fluid (100m mol/L Tris-Cl, 10m mol/L EDTA, 4 ℃ of storages), 10% (w/v) sodium lauryl sulphate (SDS), 5mol/L NaCl, LB (Luria-Bertani) substratum (4 ℃ of storages), mentioned reagent all needs autoclaving; 20mg/mL Proteinase K (be divided into the aliquot that single uses and be stored in-20 ℃), 24: 1 chloroform/primary isoamyl alcohol, 25: 24: 1 phenol/chloroform/primary isoamyl alcohol, Virahol, 70% ethanol
2 equipment and equipment: PTC-100 type PCR instrument, PB-20 type pH meter, ME 215S electronic balance, the TGL-15G high speed tabletop centrifuge, 1 of biochemical incubator, 1 of OD260 and OD280 ultraviolet spectrophotometer, 1 in refrigerator, 1 of water-bath, 1 of autoclave sterilizer, 1 of the adjustable liquid-transfering gun of 1000 μ L, 1 of the adjustable liquid-transfering gun of 200 μ L, 1 of the adjustable liquid-transfering gun of 10 μ L, 10 μ L, 200 μ L and 5000 μ L liquid-transfering gun heads are some, 20 in culture dish, disposable plastic gloves is some, EP pipe and PCR pipe are some, 1 in 20L acid cylinder
3 vessel treatment processs:
All glasswares all soak more than 6 hours with the potassium bichromate washing lotion before use, rinse well with tap water, distilled water, ultrapure water successively then, dry, and it is standby to sterilize then.EP pipe and PCR pipe etc. are disposable use, and be standby behind the autoclave sterilization.
4 operation stepss:
Get strain intestinal bacteria and in the LB substratum, be cultured to state of saturation, the culture of getting 1.0mL in the EP pipe, the centrifugal 2min of 12000 commentaries on classics/min.
Abandon supernatant, throw out adds the TE damping fluid of 567 μ L, blows and beats repeatedly with suction pipe and makes it resuspended.Add the SDS of 30 μ L10% and the Proteinase K of 3 μ L 20mg/mL, mixing is in 37 ℃ of incubation 45min～1h.
Chloroform/primary isoamyl alcohol of 600 μ L such as adding, mixing, centrifugal 5 minutes of 12000 commentaries on classics/min change supernatant liquor in the new EP pipe over to.
Add isopyknic phenol/chloroform/primary isoamyl alcohol, mixing, centrifugal 5 minutes of 12000 commentaries on classics/min change supernatant liquor in the new EP pipe over to.
Add 0.6 volume Virahol, mix up to DNA precipitating gently, centrifugal 2 minutes of 5000 commentaries on classics/min.
Abandon supernatant, throw out dries with 70% washing with alcohol of 1mL.
With precipitating the TE damping fluid that heavily is dissolved in 100 μ L, shake up.
Measure the OD value of the genomic dna template of extracting with ultraviolet spectrophotometer, thereby calculate its DNA concentration.Method of calculation are:
DNA concentration (μ g/ μ L)=OD value 260nm * diluted sample multiple * 50/1000
The genomic dna template is carried out mark, store under 4 ℃ of conditions, standby.
The genomic dna that extracts microorganism as stated above carries out pcr amplification as template DNA.
The 3rd step: pcr amplification:
50 μ L reaction systems:
10 * PCR damping fluid, 5 μ L
Tag enzyme 2U/ μ L 1 μ L
MgCl2(25mM) 5μL
dNTP (2.5mM) 5μL
ddH20 29μL
Upstream primer 10 μ M 2 μ L
Upstream primer 10 μ M 2 μ L
Template DNA 1.5 μ g/ μ L 1 μ L
Polymerase chain reaction in present method applied molecular biology, the reaction primer:
Upstream primer: 5 '-tgt tca gtg gca aga gtt-3 '
Downstream primer: 5 '-taa tcg ata tac ccg ctc-3 '
The 3rd step: product is observed:
1 required reagent and equipment: dna molecular amount standard (DNA marker:800bp, 700bp, 600bp, 500bp, 400bp, 300bp), agar Icing Sugar, ethidium bromide (10mg/mL) solution, Tris, EDTA, glacial acetic acid, computer use electrophoresis apparatus, rubber moulding, microwave oven, 2 of 1000mL vials, 1 of 500mL ground Erlenmeyer flask, 1 in 1000mL graduated cylinder, disposable glove some more;
The preparation of 2 gels.Configuration 1.5% agarose solution heats in microwave oven earlier, and it is fully dissolved, and particulate matter is not arranged, in order to avoid influence the observation of product.In 2.0 μ L ethidium bromide solutions: the ratio of 15mL agarose solution adds ethidium bromide in agarose solution.The effect of ethidium bromide is to make the DNA of amplification dyeing, so that observe under ultraviolet lamp.Ethidium bromide has severe toxicity, so the operation of all contact ethidium bromides must be with gloves.
The configuration of 3 TAE electrophoresis liquid.Weighing 4.84g Tris, 2mL0.5mol/LEDTA, 1.14mL glacial acetic acid, constant volume generally need configuration 2000mL electrophoresis liquid to 1000mL.
4 fall glue.The agarose solution that configures is poured in the offset plate, treat that it solidifies after, put into electrophoresis chamber.
5 electrophoresis.PCR product that amplification is good and damping fluid are pressed 1: 1 mixed, join in the gel well, add dna molecular amount standard (Marker carries in the test kit) simultaneously, connect power supply, setting voltage and electric current, beginning electrophoresis.
6 observe.Amplified production is observed pictures taken under UV-light.
The applicant utilizes the inventive method, has detected 15 water samples altogether, and water sample 2-8 takes from water tap water; Water sample 9-12 takes from the storage of water in the municipal secondary pipe network retention basin, and the water storage time is about 24 hours; Water sample 13-15 takes from the clean water basin of water factory.The detected result of each water sample colibacillus PCR method as depicted in figs. 1 and 2, Fig. 1 and Fig. 2 show, detect the intestinal bacteria of tap water with the PCR method, water sample 2, the colibacillary detected result of 5-8,9-12 are all positive, and the colibacillary detected result of water sample 3-4,13-15 is negative.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410022322 CN1286986C (en) | 2004-04-14 | 2004-04-14 | Method for fast detecting colibacillus in drinking water |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410022322 CN1286986C (en) | 2004-04-14 | 2004-04-14 | Method for fast detecting colibacillus in drinking water |
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| Publication Number | Publication Date |
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| CN1563422A true CN1563422A (en) | 2005-01-12 |
| CN1286986C CN1286986C (en) | 2006-11-29 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100427598C (en) * | 2006-06-12 | 2008-10-22 | 南京大学 | Extraction of Microbial Total DNA and Identification of Biomass in Lake Sediments |
| CN102382818A (en) * | 2011-11-01 | 2012-03-21 | 南京大学 | Method for quickly and efficiently extracting total DNA in drinking water |
| CN106680474A (en) * | 2017-02-04 | 2017-05-17 | 上海为然环保科技有限公司 | Instrument capable of quickly detecting escherichia coli on household tableware |
| CN107589117A (en) * | 2017-08-22 | 2018-01-16 | 贵州省畜牧兽医研究所 | A kind of quick microscopy diagnostic kit of swine eperythrozoonosis and preparation method thereof |
-
2004
- 2004-04-14 CN CN 200410022322 patent/CN1286986C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100427598C (en) * | 2006-06-12 | 2008-10-22 | 南京大学 | Extraction of Microbial Total DNA and Identification of Biomass in Lake Sediments |
| CN102382818A (en) * | 2011-11-01 | 2012-03-21 | 南京大学 | Method for quickly and efficiently extracting total DNA in drinking water |
| CN106680474A (en) * | 2017-02-04 | 2017-05-17 | 上海为然环保科技有限公司 | Instrument capable of quickly detecting escherichia coli on household tableware |
| CN107589117A (en) * | 2017-08-22 | 2018-01-16 | 贵州省畜牧兽医研究所 | A kind of quick microscopy diagnostic kit of swine eperythrozoonosis and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN1286986C (en) | 2006-11-29 |
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