CN1563422A - Method for fast detecting colibacillus in drinking water - Google Patents

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

The quick detection method of intestinal bacteria in the tap water

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.

Claims (1)

1, the quick detection method of intestinal bacteria in the tap water is characterized in that may further comprise the steps:

(1) extraction of collection of microorganism and DNA thereof 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) extraction of microbial DNA in the water sample:

A, in the 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 0D 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

I, the genomic dna template is carried out mark, store, standby;

(3) pcr amplification;

The reaction primer:

Upstream primer: 5 '-tgt tca gtg gca aga gtt-3 '

Downstream primer: 5 '-taa tcg a ta tac ccg ctc-3 '

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.