CN102435745A - Kit used for rapidly detecting Escherichia coli O157:H7 in sample, and detection method thereof - Google Patents

Abstract

The invention relates to a kit used for rapidly detecting Escherichia coli O157:H7 in a sample, and a detection method thereof. The invention belongs to the technical field of immunological detection. According to the invention, a heated and deactivated Escherichia coli O157:H7 immunogen is used for immunizing a healthy New Zealand rabbit, such that a polyclonal antibody is obtained, and the polyclonal antibody is adopted as a coating antibody; a BALB/C mouse is immunized, and cell fusion is carried out, such that a monoclonal antibody is obtained, and the monoclonal antibody is adopted as a secondary antibody; and a double-antibody sandwich ELISA kit of Escherichia coli O157:H7 in foodstuffs (meat) is established. With the kit, a rapid and highly efficient detection means is provided for the detection of the residue of Escherichia coli O157:H7 in foodstuffs. The kit is advantaged in relatively low cost, relatively good stability, and relatively good repeatability. According to the invention, a detection limit is 105cfu/mL. The kit and the method are suitable for large-batch detections of samples.

Description

Kit and the detection method of Escherichia coli O157: H7 in a kind of fast detecting sample

Technical field

Kit and the detection method of Escherichia coli O157: H7 belong to the immunology detection technical field in a kind of fast detecting sample.

Background technology

Escherichia coli O157: H7 is a kind of important zoonosis pathogenic bacteria; This bacterium is widespread in nature; Often cause serious diarrhoea and septicemia; Serious caused hemolytic uremic syndrome (HUS), thrombocytopenic purpura,thrombotic (TTP), wherein concurrent HUS and TTP person's mortality ratio are up to about 80%, and harm is very serious.

Big quantity research shows: animals such as ox, pig, fowl all can be colibacillary natural reservoir (of bird flu viruses); This bacterium in animal body can long-term existence and continuous outside discharge of bacteria; Pollute the trunk of surrounding enviroment and animal; Cause the animal sources humans and animals that higher bacterial bearing rate is all arranged, food receives the chance of its pollution a lot.Infection how to control O157:H7 is the important topic in the present public hygienics, thus to Escherichia coli O157: H7 fast, accurately check is the necessary means that guarantees human health.

The method of traditional bacterial detection has comprised biochemical cultivation, isolation identification, and the check program complicacy is loaded down with trivial details, the report assay roughly needs 4～7d, and is consuming time oversize, and detection sensitivity is low.So, set up the key problem that the rapid and precise detection method is the pathogenic microorganism Inspection Research always.Immunoassay with its accuracy, specificity, characteristics such as the scope of application is wide, detection speed is fast and expense is low, becomes one of method of widespread use in the check.

Summary of the invention

The purpose of this invention is to provide the kit of Escherichia coli O157: H7 in a kind of fast detecting sample,, fast, the Escherichia coli O157 in the test sample: H7 delicately, especially be fit to the mass detection of various samples so that can be easy

Technical scheme of the present invention: utilize heat-killed Escherichia coli O157: H7 (ATCC 35150) to obtain polyclonal antibody as coated antibody as the healthy new zealand white rabbit of immunogen immune; Immune balb/c mice also carries out Fusion of Cells to obtain monoclonal antibody anti-as two, has set up the kit of the double antibodies sandwich ELISA method of Escherichia coli O157: H7 in the food.Escherichia coli O157: H7 has been that following document is open, document 1: [Food Microbiology 23 (2006) 162 – 168.The use of Fourier transform infrared spectroscopy to differentiate Escherichia coli O157:H7 from other bacteria inoculated into apple juice; Murad A. Al-Holy ^a, Mengshi Lin ^b, Anna G. Cavinato ^c, Barbara A. Rasco ^b];

Document 2: [Food Research International 39 (2006) 98 – 105.Inactivation of food spoilage bacteria and Escherichia coli O157:H7 in phosphate buffer and orange juice using dynamic high pressure; Imane Tahiri, Joseph Makhlouf *, Paul Paquin, Ismail Fliss].

Kit of the present invention consists of the following components:

(1) reaction plate: the polyclonal antibody with the dilution of the carbonate buffer solution of 0.05M encapsulates in the 96 hole ELISA Plates every hole 100 μ L.4 ℃ of incubated overnight are according to conventional ELISA method sealing washing.

(2) Escherichia coli O157: H7 positive control standard items and negative control standard items.

(3) the monoclonal antibody antiserum that dilutes with antibody diluent.

(4) with the sheep anti mouse (GAM-HRP) of antibody diluent dilution horseradish peroxidase-labeled.

(5) colour developing liquid A, colour developing liquid B, before using with A and B with the 5:1 volume mixture.

(6) stop buffer (2M sulfuric acid solution).

More detailed step is:

Main solution preparation

1) preparation phosphate 0.01M (PBS) damping fluid:

Na ₂HPO ₄·12H ₂O 3.62?g

KH ₂PO ₄ 0.2?g

NaCl 0.2g

KCl 8.0g

Add ultrapure water and be diluted to 1000 mL.

2) preparation carbonate 0.05M, pH9.6 (CBS) buffer solution:

Na ₂CO _3? 1.59g

NaHCO ₃ 2.93g

Add ultrapure water and be diluted to 1000 mL.

3) preparation PBST solution: the PBS solution that contains 0.05% Tween – 20.

4) preparation confining liquid: what contain 0.1% gelatin encapsulates damping fluid (0.01M PBS damping fluid).

5) preparation antibody diluent: the PBST solution that contains 0.1% gelatin.

6) colour developing liquid:

A liquid: 0.933 g citric acid, 3.68 g Na ₂HPO ₄12H ₂O, 18 μ L 30%H ₂O ₂, be settled to 100 mL with ultrapure water.

B liquid: 60 mg 3,3 ^/, 5,5 ^/-tetramethyl benzidine (TMB) is dissolved in the 100 mL monoethylene glycol;

Before using with A and B with the 5:1 volume mixture.

7) H of stop buffer: 2M ₂SO ₄

Detection kit provided by the present invention uses step following:

Prepare PBST solution (0. 01 mol/L pH7. 4,0.15mo1/L NaCl, 0. 5%Tween-20) in advance.

A, in reaction plate, add testing sample, positive criteria article, negative standard items respectively, 100 μ L/holes are in 37 ℃ of incubation 1h.

B, washing: with PBST washing reaction plate 3-5 time, each 3min, 220 μ L/ holes dry reaction plate then.

The monoclonal antibody antiserum that c, adding are diluted with antibody diluent, 100 μ L/holes, 37 ℃ of reaction 1h.

D, washing: with PBST washing reaction plate 3-5 time, each 3min, 220 μ L/ holes dry reaction plate then.

E, add ELIAS secondary antibody (sheep anti mouse GAM-HRP), 100 μ L/holes, 37 C react 1h.

F, washing: with PBST washing reaction plate 3-5 time, each 3min, 220 μ L/ holes dry reaction plate then.

G, colour developing: add colour developing liquid 100 μ L/holes, colour developing 15min.

H, termination: add stop buffer 100 μ L/holes.

I, mensuration: detect OD with ELIASA _450nm, i.e. light absorption value A450.

When [(the light absorption value A450 of sample)/(negative standard items light absorption value A450)]＞2.1, and [(positive criteria article light absorption value A450)/(negative standard items light absorption value A450)]＞2.1 o'clock, testing sample is positive;

When [(the light absorption value A450 of sample)/(negative standard items light absorption value A450)]＜2.1, and [(positive criteria article light absorption value A450)/(negative standard items light absorption value A450)]＞2.1 o'clock, testing sample is negative.

Beneficial effect of the present invention: the Escherichia coli O157 that the present invention sets up: the double antibodies sandwich ELISA detection method of H7, to the Escherichia coli O157 in the food: H7 detects, and detection sensitivity is 10 ⁵Cfu/mL.In 4h, can accomplish detection, can quick and precisely accomplish detection, be particularly suitable for the mass detection of sample.

Specific embodiments

Below further specify the present invention through embodiment.

The preparation of embodiment 1. immunogenes and positive criteria article

Escherichia coli O157: H7 (ATCC 35150) is seeded on sorbierite Mai Kangkai (SMAC) flat board; Cultivate 24h for 37 ℃; Picking list bacterium colony increases bacterial context soup in improvement E.C ovobiocin, 37 ℃, 200r/min shaken cultivation 17h, counting; Boiling water bath heats deactivation at 1 night, physiological saline adjustment Escherichia coli O157: H7 (ATCC 35150) concentration to 5 * 10 ⁹Cfu/mL is immunogene; Using PBS damping fluid adjustment concentration is 10 ⁷The positive reference standards of cfu/mL, the negative reference standards of PBS damping fluid.

Embodiment 2. encapsulates sero-fast preparation

1) animal used as test: the healthy new zealand white rabbit that to select 32 monthly ages, body weight be 1.5 –, 2 kg is an animal used as test.

2) emulsification: with complete with equivalent or the incomplete freund adjuvant of 0.5mL immunogene with mixing paddling process with its emulsification, until an emulsion is splashed in the water, is not scattered float on the water surface till.

3) immunization method: initial immunity reagent that emulsification is good is multi-point injection in the rabbit back, and 1 mL/ only.Immunity behind the initial immunity is called booster immunization, and booster immunization is used freund 's incomplete adjuvant emulsification, and booster immunization is intramuscular injection, per two all booster immunizations once, dosage is identical with initial immunity.

4) blood sampling: from ear edge vein exploitating blood, adopt indirect non-competing ELISA to measure antiserum titre behind 3 booster immunizations.Wait to tire reach requirement (1 ︰ 128000) after, adopt the ear vein bloodletting acquisition antiserum that combines with the heart bloodletting, be collected in 50 mL and sterilize in the plastic centrifuge tube.

5) purifying antibody and preservation: antiserum X mL is diluted to 2X mL with the physiological saline of equivalent, under agitation dropwise adds the saturated ammonium sulfate with dilution back serum equivalent (2X) then, place for 4 ℃ and it was fully precipitated in 3 hours.The centrifugal 20min of 3000r/min abandons supernatant, is precipitated to X mL with physiological saline solution, drips saturated ammonium sulfate (X/2) mL more gradually.Placing for 4 ℃ fully precipitated it in 3 hours; Repeat the above-mentioned second step process 1 time; The centrifugal back of last gained sediment is dissolved to X mL with 0.02mol/L PBS (pH 7.4), and 0.02mol/L PBS in the bag filter of packing into (pH 7.4) is dialysis fully, during change liquid 3 times; Having dialysed concentrates, and is diluted to 3 μ g/mL as polyclonal antiserum with encapsulating damping fluid.

3. the sero-fast preparation of monoclonal

1) animal used as test: select 38 ages in week, about body weight 20g, female BALB/C mice is animal used as test.

2) immunization method: every mouse peritoneal injection 0.2mL immunogene, 2 weeks of every interval with same dosage booster shots once.

3) blood sampling: from the tail vein blood sampling, adopt indirect non-competing ELISA to measure antiserum titre behind 3 booster immunizations.Wait to tire and no longer rise, lumbar injection is measured immunogene equally, carries out Fusion of Cells by conventional method after 3 days.

4) Fusion of Cells: get immune mouse spleen cell and SP2/0 myeloma cell conventional fusion the under 50% PEG effect, be inoculated in 96 well culture plates respectively, place 37 ℃, 5% CO ₂Incubator is cultivated.

5) filtering hybridoma: adopt indirect non-competing ELISA, the hybridoma in screening strong positive hole is transferred to 24 well culture plates with it.

6) clone cultivates and Antibody Preparation: carry out cloning with limiting dilution assay and cultivate.When cell grow to be paved with the hole at the bottom of 1/10 the time, use again with quadrat method and detect, the strong positive hole is cloned again, 3-4 time so repeatedly, reach 100 % until positive rate.With the hybridoma enlarged culture, be injected in through the pretreated BALB/C mice of paraffinum liquidum abdominal cavity, every 2 * 10 ⁶Individual hybridoma, 7～10 days mouse web portion protuberances, living body puncture extracts ascites.With sad-ammonium sulfate method antibody purification from mouse ascites, be diluted to 1 μ g/mL as monoclonal anti serum with antibody diluent.

3, artificial contamination's sample double antibodies sandwich ELISA course of reaction:

To encapsulate serum and encapsulate 96 hole ELISA Plates with encapsulating damping fluid as serial dilution, 100 μ L/ holes are in 4 ℃ of refrigerator overnight.Take out ELISA Plate and be back to room temperature next day, and 200 μ L PBST solution are injected in every hole, and vibration 3 min firmly get rid of cleansing solution on the shaking table, do at the thieving paper arsis, continue washing 2 times.Following washing methods is identical.

Fully after the washing, with sealing damping fluid sealase target, 200 μ L/ holes are taken out oven dry and are the good reaction plate of sealing in the kit behind incubation 2 h in 37 ℃ of incubation casees.

1) getting 25g pork is representative sample, puts into sterilization 500 ml homogeneous cups, adds a small amount of improvement E.C ovobiocin and increases the aseptic homogenate that bacterial context soup carries out strictness, at last quantitatively to 225 ml.Inoculate the Escherichia coli O157 that 100 μ L have used 10 times of dilutions of 0.9% physiological saline respectively: H7 bacterium liquid, 37 ℃, 200r/min shaken cultivation 17h, inoculation counting agar plate is counted, and confirms Escherichia coli O157 in the sample: H7 content.

2) in the differential responses plate, add testing sample, positive criteria article, the negative standard items of variable concentrations respectively, 100 μ L/holes are in 37 ℃ of incubation 1h.

3) add the monoclonal antibody antiserum that dilutes with antibody diluent, 100 μ L/ holes, washing behind 37 ℃ of reaction 1h, bat are done.

4) add ELIAS secondary antibody (sheep anti mouse GAM-HRP), 100 μ L/holes, washing behind the 37 C reaction 1h, bat are done.

5) add colour developing liquid (TMB and substrate solution ratio are 1:5) 100 μ L/holes, the 37 ℃ of reactions in dark place, 15 min take out every hole, back and add 100 μ L stop buffers (sulfuric acid of 2 mol/L), measure light absorption value A450 with ELIASA.

When [(the light absorption value A450 of sample)/(negative standard items light absorption value A450)]＞2.1, and [(positive criteria article light absorption value A450)/(negative standard items light absorption value A450)]＞2.1 o'clock, explain that testing sample is positive; [(the light absorption value A450 of sample)/(negative standard items light absorption value A450)]＜2.1, and [(positive criteria article light absorption value A450)/(negative standard items light absorption value A450)]＞2.1 o'clock, explain that testing sample is negative.The sample of variable concentrations is detected, and the result shows that concentration is 10 ⁵During cfu/mL, reaction result is positive, and concentration is 10 ⁴During cfu/mL, reaction result is negative, detects and is limited to 10 ⁵Cfu/mL.

4, The specificity

With Escherichia coli O157: H7, salmonella, vibrio parahaemolytious, Shigella bogdii, Listeria monocytogenes bacterium liquid (concentration 10 ⁷Cfu/mL) as testing sample, carry out cross reaction research, this kit reaction result is all negative.

Claims (3)

1. the double antibodies sandwich enzyme-linked immunologic detecting kit of Escherichia coli O157 a: H7; It is characterized in that utilizing heat-killed Escherichia coli O157: the healthy new zealand white rabbit of H7 immunogen immune obtains polyclonal antibody as coated antibody; Immune balb/c mice also carries out Fusion of Cells to obtain monoclonal antibody anti-as two, has set up the kit of the double antibodies sandwich ELISA of Escherichia coli O157: H7; Escherichia coli O157: H7 is ATCC 35150, and said kit consists of the following components:

(1) reaction plate: the polyclonal antibody with the dilution of the carbonate buffer solution of 0.05M encapsulates 96 hole ELISA Plates, every hole 100 μ L; 4 ℃ of incubated overnight according to conventional ELISA method sealing washing, are the reaction plate in the kit;

(2) Escherichia coli O157: H7 positive control standard items; The negative control standard items;

Escherichia coli O157: H7 is seeded on the sorbierite Mai Kangkai SMAC flat board; Cultivate 24h for 37 ℃; Picking list colony inoculation increases 37 ℃ of bacterial context soup, 200r/min shaken cultivation 17h in improvement E.C ovobiocin; Counting, boiling water bath heats deactivation at 1 night, physiological saline adjustment Escherichia coli O157: H7 concentration to 5 * 10 ⁹Cfu/mL is immunogene;

Adjust Escherichia coli O157 with sterilization PBS damping fluid: H7 concentration is 10 ⁷The positive reference standards of cfu/mL, the negative reference standards of sterilization PBS damping fluid;

(3) the monoclonal antibody antiserum that dilutes with antibody diluent;

Antibody diluent: the PBST solution that contains 0.1% gelatin;

(4) ELIAS secondary antibody: with the sheep anti mouse GAM-HRP of the horseradish peroxidase-labeled of antibody diluent dilution;

(5) colour developing liquid A:0.933 g citric acid, 3.68 g Na ₂HPO ₄12H ₂O, 18 μ L 30%H ₂O ₂Be settled to 100 mL with ultrapure water;

Colour developing liquid B:60 mg 3,3 ^/, 5,5 ^/-tetramethyl benzidine TMB is dissolved in the 100 mL monoethylene glycol;

Before using with A and B with the 5:1 volume mixture;

(6) stop buffer: 2M sulfuric acid solution.

2. the double antibodies sandwich enzyme-linked immunologic detecting kit of the described Escherichia coli O157 of claim 1: H7 is characterized in that in the step (1) that being diluted to polyclonal antibody concentration is 3 μ g/mL with the polyclonal antibody of the carbonate buffer solution dilution of 0.05M; With the monoclonal antibody antiserum of antibody diluent dilution, being diluted to the monoclonal anti bulk concentration is 1 μ g/mL in the step (3).

3. the detection method of the double antibodies sandwich enzyme-linked immunologic detecting kit of the described Escherichia coli O157 of claim 1: H7 is characterized in that step is following:

Prepare in advance in PBST solution: pH7.4, the 0.01mol/L PBS solution and contain 0.15mo1/L NaCl and 0.5% Tween-20;

A, in reaction plate, add testing sample, positive control standard items, negative control standard items respectively, 100 μ L/holes are in 37 ℃ of incubation 1h;

B, washing: with PBST washing reaction plate 3-5 time, each 3min, 220 μ L/ holes dry reaction plate then;

The monoclonal antibody antiserum that c, adding are diluted with antibody diluent, 100 μ L/ holes, 37 ℃ of reaction 1h;

D, washing: with PBST washing reaction plate 3-5 time, each 3min, 220 μ L/ holes dry reaction plate then;

E, add ELIAS secondary antibody: GAM-HRP, 100 μ L/holes, 37 C react 1h;

F, washing: with PBST washing reaction plate 3-5 time, each 3min, 220 μ L/ holes dry reaction plate then;

G, colour developing: add colour developing liquid 100 μ L/holes, lucifuge colour developing 15min;

H, termination: add stop buffer 100 μ L/holes;

I, mensuration: survey light absorption value OD, i.e. light absorption value A450 with ELIASA 450nm;

When [(the light absorption value A450 of sample)/(negative standard items light absorption value A450)]＞2.1, and [(positive criteria article light absorption value A450)/(negative standard items light absorption value A450)]＞2.1 o'clock, testing sample is positive;

When [(the light absorption value A450 of sample)/(negative standard items light absorption value A450)]＜2.1, and [(positive criteria article light absorption value A450)/(negative standard items light absorption value A450)]＞2.1 o'clock, testing sample is negative.