CN103439496A - Escherichia coli O157:H7 enrichment and rapid detection method - Google Patents
Escherichia coli O157:H7 enrichment and rapid detection method Download PDFInfo
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Abstract
The invention applies difunctional nanogold magnetic particles and provides an escherichia coli O157:H7 rapid detection method integrating an immunomagnetic bead capture technique and an immunochromatography technique. The immune magnetic separation and immunochromatography are organically integrated, the step of eluting the escherichia coli O157:H7 from an immunomagnetic bead is eliminated, as a result, the capture efficiency is improved; the step of spraying colloidal gold on a binding pad is eliminated, so that the immunological reaction is much evener, and the variable coefficient is small during quantitative determination; the workload and the living contaminant rate are reduced. The basic train of thought of the detection method is exploring the effective combination method of the nanogold magnetic particles and an antibody, optimizing the condition of enriching the escherichia coli O157:H7 on the nanogold magnetic particles and the antibody, and conducting rapid quantitative detection by taking the immunochromatography technique as a carrier and a double-antibody sandwich as a detection principle.
Description
Technical field
The present invention relates to the microorganism detection field, specifically adopt difunctional nanometer gold magnetic particle integrated immune magnetic capture technology and immunochromatography fast detecting Escherichia coli O 157: H7.
Technical background
The food origin disease that microorganism causes is the No.1 food-safety problem of whole world ,Ye Shi China.In food-borne pathogens, Escherichia coli O 157: H7 is the most dangerous one, and its infective dose is very low, and minimum 10 viable bacterias are caused a disease with regard to PI.Cramp and non hemorrhagic diarrhoea generally first appear in Escherichia coli O 157: H7 after infecting, the patient over 70% can develop into hemorrhagic diarrhea, and 30%～60% case has the vomiting phenomenon, and 30% case has low heating paresthesia.The patient of 3%-5% can develop into hemolytic uremic syndrome and cause acute renal failure and death.Can pollute pork, poultry, beef, milk, fruit juice, cold sandwich, vegetables and drinking-water etc., it is because eaten by the food of this fungi pollution or the potable water of strict sterilization not mostly that this bacterium popular breaks out.In addition, can also be by interpersonal contact transmission.
At present, the method for detection Escherichia coli O 157: H7 has tradition to separate identification method, PCR method, method for biosensor, immunological method etc.Separate identification method for tradition and detect Escherichia coli, much time power (5-7 days), need the professional to operate, therefore the method can only be analyzed the cause of poisoning for law enforcement agency, and the market security status of food is assessed, the quality safety of the front product that dispatches from the factory is made to the confirmation of quick effective evaluation and acute poisoning event reason and can't meet modern enterprise.PCR method and method for biosensor all have higher detection sensitivity, generally can be reduced to 24-48 hour detection time (according to different test items), but the method requires to have more high-quality operating personnel, detecting instrument and testing conditions preferably, therefore more difficult in basic unit and enterprise's spread use, still be difficult to meet enterprise detection time simultaneously the front Product quality and safety that dispatches from the factory is carried out to the rapid-action actual demand, and the homology of pathogenic bacteria and DNA fragmentation can't be differentiated extremely/be lived to the method, easily cause the higher result of false positive, and, when detecting a lot of food substrate, can suppress because of the existence of some materials again the activity of archaeal dna polymerase, thereby cause false negative.Immunology is immune chromatography method especially, does not need complex instrument equipment, and detection time, fast (general 15 min can obtain result), easy to operate, but the overall detection sensitivity of these class methods is on the low side at present, (needs bacterial concentration need reach 10
5-10
6cFU/mL), therefore for food samples, detect, often need the bacterium time that increases of growing.Current Escherichia coli O 157: the H7 immunological method adopts the colloidal gold immunochromatographimethod technology for detection, from increasing bacterium to detecting the detection that needs could realize at least 24 hours single bacterium 25 g samples.
Because microbial growth has its specific physiological period, shorten chronergy by optimum culture condition not obvious; Therefore many employing immunomagnetic beads isolation technics enrichment.The immunomagnetic beads isolation technics that depends on specific antibody can improve the detection sensitivity of microorganism to be measured effectively, become gradually one of food-borne pathogens specific isolation and concentrated effective means, and be widely used in Escherichia coli O 157: the enrichment of the food-borne pathogens such as H7, greatly shortened the food-borne pathogens whole detection time.
The colloidal gold immunity chromatography that the film of take is solid phase carrier is the novel detection method grown up on monoclonal antibody technique, colloid gold immune technology and new material technology basis the eighties in 20th century.Because it is quick, convenient, do not need specific installation, the result judgement is directly perceived, can be detected at scene, more and more is subject to people's attention in recent years, its technical development is rapid, detection field be widely applied.But for some antigen or the extremely low sample of antibody content, the very light judged result that almost with the naked eye is difficult to of the color of label, although the instrument for the immuno-chromatographic test paper strip interpretation of the marks such as collaurum is arranged in the market, but this instrument only can be to the label color detection at surface of solid phase carriers, and the label of solid phase carrier inside is difficult to detect, the sensitivity therefore detected is very limited.May contain result and the sensitivity that colour substance can have a strong impact on detection in the biological specimen simultaneously detected.
Summary of the invention
The object of the invention is to provide a kind of quick, sensitive, easy Escherichia coli O 157: H7 qualitative and quantitative analysis technology.
Concrete scheme of the present invention is as follows:
Escherichia coli O 157: H7 enrichment and method for quick comprise the following steps: the gold-magnetic particles that 1) prepares the coupling monoclonal antibody; Getting Chinese People's Anti-Japanese Military and Political College's enterobacteria O157:H7 monoclonal antibody mixes with nanometer gold magnetic particle; When 37 ℃ of temperature, on the gyroscope that to be placed on rotating speed be 10-15rpm, coupling time 30-60min, magnetic separates 3-5min, abandons supernatant; Clean after 3 times with cleaning buffer solution, with the 1mL sealer, mix sealing 1h with magnetic bead; 2) use the gold-magnetic particles of coupling monoclonal antibody to catch bacterium: to cultivate Escherichia coli O 157: H7, bacterial concentration is adjusted into to 10
7cFU/mL, 10
6cFU/mL, 10
5cFU/mL, 10
4cFU/mL, respectively get 1 mL standby; Get testing sample solution 1 mL, each concentration bacterium liquid 1 mL, respectively with the gold-magnetic particles 100-150 μ g of step 1) gained coupling monoclonal antibody, 37 ℃ of temperature, rotating speed 10-15rpm mixes hatches 30-60min; After hatching rear magnetic separation 3-5min, abandon supernatant, with after the PBS buffer solution for cleaning, redissolve in PBS; 3) make immuno-chromatographic test paper strip; Using Escherichia coli O 157: how anti-and anti-being sprayed onto on nitrocellulose membrane respectively as detection line T line and nature controlling line C line of the anti-mouse two of rabbit of H7 rabbit, concentration is 1.0-2.0mg/mL, discharge rate is 0.5-1.0 μ L/cm, 37 ℃ of vacuum drying are spent the night, sample pad, nitrocellulose membrane, thieving paper, filter paper are sticked on the PVC base plate successively, be cut into the wide strip of 4mm after posting, be installed; The test strips prepared is placed in to Fresco Bag and adds the drying agent sealing, be placed in dry cylinder and save backup; 4) utilize the double antibodies sandwich method to be measured sample; The gold-magnetic particles that captures bacterium is diluted to concentration 50-150 μ g/mL, gets 100 μ L and be added drop-wise in the test strips well, read with the immunochromatographiassays assays instrument after 10min, record the value of T line absorbance, C line absorbance and T/C; 5) qualitative analysis: the result that detects by an unaided eye is carried out qualitative analysis, and the T line develops the color has Escherichia coli O 157: H7 in interpret sample, and the T line does not develop the color not to be had Escherichia coli O 157: H7 or contain Escherichia coli O 157 in interpret sample: the amount of H7 is lower than 10
4cFU/mL; 6) quantitative test: use the immunochromatographiassays assays instrument to measure the absorbance of T line, C line, the ratio of the absorbance of T line and C line is designated as the T/C value, and the concentration of different bacterium of take is horizontal ordinate, and the T/C value is ordinate drawing standard curve; With reference to the canonical plotting of doing, determine the Escherichia coli O 157 in common sample: H7 quantity.
Step 1) nanometer gold magnetic particle particle diameter is 50nm; The amount of the nanometer gold magnetic particle of the corresponding 50nm of the amount 200-300 μ g of the enterobacteria O157:H7 of step 1) Chinese People's Anti-Japanese Military and Political College monoclonal antibody is 0.5-1.0mg.
The described immuno-chromatographic test paper strip of step 3) is to paste successively sample pad, nitrocellulose membrane, thieving paper, filter paper composition on adhesive base, there is no pad.
Use Escherichia coli O 157: the H7 colloidal gold strip, the method of simultaneously using the immunochromatographiassays assays instrument quantitatively to detect, it is characterized in that: the Escherichia coli O 157 of preparing known series concentration: H7 solution, measure the numerical value of its corresponding optical density with the immunochromatographiassays assays instrument, according to this series of values and corresponding concentration Criterion curve, then will detect the test strips of sample and put into the immunochromatographiassays assays instrument, according to the numerical value of immunochromatographiassays assays instrument output, look into the content that canonical plotting can draw Escherichia coli O 157 in sample: H7.
The present invention has following advantage:
1) nanometer gold magnetic particle that the present invention adopts, have the superparamagnetism of magnetic nano-particle and the performance of the surperficial efficient coupling antibody of collaurum concurrently.Due to nanometer gold magnetic particle can be surperficial by it the non-covalent bond effects such as electrostatic interaction, hydrophobic effect and Au-SH effect, the protein substances such as the absorption antibody of physical property, the coupling rate is high, and coupling effect is stable and can guarantee that in coupling process the activity of antibody is unaffected.
2) the present invention separates immune magnetic and the immunochromatography organic combination, has removed from Escherichia coli O 157: the step that H7 elutes from immunomagnetic beads, improved capture rate; Removed from collaurum is sprayed on to the step on pad, immunological response is homogeneous more, and while quantitatively detecting, the coefficient of variation is little; Workload and living contaminants probability have been reduced.
3) can carry out qualitative and quantitative detection to object Escherichia coli O 157: H7 simultaneously.
The accompanying drawing explanation
Fig. 1 is the structural drawing of nanometer gold magnetic particle coupling antibody
Fig. 2 is the process flow diagram of nanometer gold magnetic particle immunity magnetic separating Escherichia coli O157:H7
Fig. 3 is used immuno-chromatographic test paper strip to detect the schematic diagram of negative sample
Fig. 4 is used immuno-chromatographic test paper strip to detect the schematic diagram of positive sample.
Embodiment
The present invention utilizes the magnetic property of nanometer gold magnetic particle, characteristic color and the characteristic that is easy to coupling antibody, by the enterobacteria O157:H7 of nanometer gold magnetic particle mark Chinese People's Anti-Japanese Military and Political College antibody, with then this label being joined in sample, simultaneously, by Escherichia coli O 157: how anti-being coated on nitrocellulose membrane of H7 rabbit form detection line, anti-being coated on nitrocellulose membrane as nature controlling line of the anti-mouse two of rabbit, the sample pad that to process again, nitrocellulose membrane, thieving paper and filter paper stick on back up pad successively, utilize double antibody sandwich method to detect in sample and whether contain Escherichia coli O 157: H7 and quantitatively detect Escherichia coli O 157: H7.Contain certain density Escherichia coli O 157 in testing sample: during H7, Escherichia coli O 157: H7 first is combined with the gold-magnetic particles labelled antibody, it is dripped in the test strips well, because the chromatography action-reaction forms antigen, (Escherichia coli O 157: H7)-antibody-nanometer gold magnetic particle antibody complex is enriched in to detect and is with, unnecessary nanometer gold magnetic particle label continues to move to control is with position, antibody generation immune response due to two anti-and nanometer gold magnetic particle marks, be enriched in again to control and be with, after 1-10 minute, take out test strips, with the naked eye detecting observations on band and quality control band, also can carry out by the immunochromatographiassays assays instrument quantitative interpretation of result.If the nature controlling line place does not all have color or there is no clear signal, illustrate that test strips is defective in quality, test invalidation.
Provide embodiment below in conjunction with technical scheme of the present invention.Following examples are the form example with concrete experimental implementation to the solution of the present invention, and experiment condition wherein and setup parameter should not be considered as the limitation to basic technical scheme of the present invention.And protection scope of the present invention is not limited to following embodiment.
Nm of gold magnetic particle is purchased from Xi'an gold magnetic nanometer biotechnology company limited, and the nanometer gold magnetic particle particle diameter is 50nm.
SkanFlexi immunochromatographiassays assays instrument is purchased from Changzhou Si Kangli bio tech ltd.
The coupling buffer compound method is as follows: after the boric acid that the borax that is 19.07g/mL by 3mL concentration is 12.37g/mL with 7mL concentration mixes, dilute 10 times.
The cleaning buffer solution compound method is as follows: take in the sterile distilled water that 0.43gMES is dissolved in 200mL, tune pH is 5.5-6.0.
The sealer compound method is as follows: get the 50mg skimmed milk power and add 1mLPBS solution to be made into sealer.
embodiment 1: use the detection of nanometer gold magnetic particle to Escherichia coli O 157 in milk: H7
1. prepare the gold-magnetic particles of coupling monoclonal antibody:
1.1 the processing of nanometer gold magnetic particle: get the coupling buffer of 200-400 μ L in the 2mL centrifuge tube, the 50nm nanometer gold magnetic particle of getting 0.5-1.0mg mixes with it, and magnetic is abandoned supernatant after separating 3-5min.
1.2 coupling reaction: get the enterobacteria O157:H7 of the Chinese People's Anti-Japanese Military and Political College monoclonal antibody 200-300 μ g prepared, mix with the nanometer gold magnetic particle 0.5-1.0mg of 50nm, be placed in the 1mL coupling buffer.When 37 ℃ of temperature, on the gyroscope that to be placed on rotating speed be 10-15rpm, coupling time 30-60min, magnetic separates 3-5min and abandons supernatant.With the 1mL cleaning buffer solution, clean 3 times.
1.3 sealing: after cleaning, with sealer 1mL, mix sealing 1h with magnetic bead.
2. use the nanometer gold magnetic particle of coupling monoclonal antibody to catch the Escherichia coli O 157 in milk: H7
After getting the sterilizing of 25mL, milk joins in the 225mL nutrient culture media, inoculates certain density Escherichia coli O 157: H7, and temperature is at 36 ℃ ± 1 ℃, and the time is that 8～18h concussion is cultivated.Bacterial concentration is adjusted into to 10
7cFU/mL, 10
6cFU/mL, 10
5cFU/mL, 10
4cFU/mL.
Get each concentration of 1mL bacterium liquid, get 1mL milk sample to be measured, add the immune nano gold-magnetic particles 100-150 μ g after sealing, mix and hatch 30-60min, 37 ℃ of temperature, rotating speed 10-15rpm.After hatching rear magnetic separation 3-5min, abandon supernatant, after cleaning with PBS, redissolve in PBS.
3. making Escherichia coli O 157: H7 immuno-chromatographic test paper strip
Using Escherichia coli O 157: how anti-and anti-being sprayed onto on nitrocellulose membrane respectively as detection line and nature controlling line of the anti-mouse two of rabbit of H7 rabbit, concentration is 1.0-2.0mg/mL, discharge rate is 0.5-1.0 μ L/cm, 37 ℃ of vacuum drying are spent the night, sample pad, nitrocellulose membrane, thieving paper are sticked on the PVC base plate successively, be cut into the wide strip of 4mm after posting, be installed.The test strips prepared is placed in to Fresco Bag and adds the drying agent sealing, be placed in dry cylinder and save backup.
4. utilize the double antibodies sandwich method sample to be estimated and used the Instrument measuring result
The gold-magnetic particles that captures bacterium is diluted to concentration 50-150 μ g/mL, getting 100 μ L is added drop-wise in the test strips well, after 10min, with the immunochromatographiassays assays instrument, read, record the value of T line absorbance, C line absorbance and T/C, the concentration of different bacterium of take is horizontal ordinate, and take respectively T line absorbance, T/C value is ordinate drawing standard curve.The result that detects by an unaided eye is carried out qualitative analysis, and the T line develops the color has Escherichia coli O 157: H7 in interpret sample, and the T line does not develop the color not to be had Escherichia coli O 157: H7 or contain Escherichia coli O 157 in interpret sample: the amount of H7 is lower than 10
4cFU/mL.
With reference to the canonical plotting of doing, determine the quantity of Escherichia coli O 157 in sample: H7.The scope of quantitative testing bacteria concentration is 10
4-10
7cFU/mL.
embodiment 2: use the detection of nanometer gold magnetic particle to Escherichia coli O 157 in beef: H7
1. prepare the gold-magnetic particles of coupling monoclonal antibody:
1.1 the processing of nanometer gold magnetic particle: get the coupling buffer of 200-400 μ L in the 2mL centrifuge tube, the 50nm nanometer gold magnetic particle of getting 0.5-1.0mg mixes with it, and magnetic is abandoned supernatant after separating 3-5min.
1.2 coupling reaction: get the enterobacteria O157:H7 of the Chinese People's Anti-Japanese Military and Political College monoclonal antibody 200-300 μ g prepared, mix with the nanometer gold magnetic particle 0.5-1.0mg of 50nm, be placed in the 1mL coupling buffer.When 37 ℃ of temperature, on the gyroscope that to be placed on rotating speed be 10-15rpm, coupling time 30-60min, magnetic separates 3-5min and abandons supernatant.With cleaning buffer solution, clean 3 times.
1.3 sealing: after cleaning, with sealer 1mL, mix sealing 1h with magnetic bead.
2. use the nanometer gold magnetic particle of coupling monoclonal antibody to catch the Escherichia coli O 157 in milk: H7
The beef meat gruel of getting 25mg joins in the 225mL nutrient culture media, mixes.Inoculate certain density Escherichia coli O 157: H7, temperature is at 36 ℃ ± 1 ℃, and the time is that 8～18h concussion is cultivated.
Bacterial concentration is adjusted into to 10
7cFU/mL, 10
6cFU/mL, 10
5cFU/mL, 10
4cFU/mL.
Get each concentration of 1mL bacterium liquid, get 1mL meat gruel sample solution to be measured, add the immune nano gold-magnetic particles 100-150 μ g after sealing, mix and hatch 30-60min, 37 ℃ of temperature, rotating speed 10-15rpm.After hatching rear magnetic separation 3-5min, abandon supernatant, after cleaning with PBS, redissolve in PBS.
3. making Escherichia coli O 157: H7 immuno-chromatographic test paper strip
Using Escherichia coli O 157: how anti-and anti-being sprayed onto on nitrocellulose membrane respectively as detection line and nature controlling line of the anti-mouse two of rabbit of H7 rabbit, concentration is 1.0-2.0mg/mL, discharge rate is 0.5-1.0 μ L/cm, 37 ℃ of vacuum drying are spent the night, sample pad, nitrocellulose membrane, thieving paper are sticked on the PVC base plate successively, be cut into the wide strip of 4mm after posting, be installed.The test strips prepared is placed in to Fresco Bag and adds the drying agent sealing.
4. utilize the double antibodies sandwich method sample to be estimated and used the Instrument measuring result
The nanometer gold magnetic particle that captures bacterium is diluted to concentration 50-150 μ g/mL, getting 100 μ L is added drop-wise in the test strips well, 10min reads with the immunochromatographiassays assays instrument, record the value of T line absorbance, C line absorbance and T/C, the concentration of different bacterium of take is horizontal ordinate, and take respectively T line absorbance, T/C value is ordinate drawing standard curve.The result that simultaneously detects by an unaided eye is carried out qualitative analysis, if the T line has color in interpret sample, bacterium to be arranged, detectability is about 10
4cFU/mL., the T line does not develop the color not to be had Escherichia coli O 157: H7 or contain Escherichia coli O 157 in interpret sample: the amount of H7 is lower than 10
4cFU/mL.
With reference to the canonical plotting of doing, determine the quantity of Escherichia coli O 157 in sample: H7.The scope of quantitative testing bacteria concentration is 10
4-10
7cFU/mL.
Claims (3)
1. Escherichia coli O 157: H7 enrichment and method for quick is characterized in that comprising the following steps: the gold-magnetic particles that 1) prepares the coupling monoclonal antibody; Getting Chinese People's Anti-Japanese Military and Political College's enterobacteria O157:H7 monoclonal antibody mixes with nanometer gold magnetic particle; When 37 ℃ of temperature, on the gyroscope that to be placed on rotating speed be 10-15rpm, coupling time 30 ~ 60min, magnetic separates 3 ~ 5min, abandons supernatant; Clean after 3 times with cleaning buffer solution, with the 1mL sealer, mix sealing 1h with magnetic bead; 2) use the gold-magnetic particles of coupling monoclonal antibody to catch bacterium: to cultivate Escherichia coli O 157: H7, bacterial concentration is adjusted into to 10
7cFU/mL, 10
6cFU/mL, 10
5cFU/mL, 10
4cFU/mL, respectively get 1 mL standby; Get testing sample solution 1 mL, each concentration bacterium liquid 1 mL, respectively with gold-magnetic particles 100 ~ 150 μ g of step 1) gained coupling monoclonal antibody, 37 ℃ of temperature, rotating speed 10-15rpm mixes hatches 30 ~ 60min; After hatching rear magnetic separation 3 ~ 5min, abandon supernatant, with after the PBS buffer solution for cleaning, redissolve in PBS; 3) make immuno-chromatographic test paper strip; Using Escherichia coli O 157: how anti-and anti-being sprayed onto on nitrocellulose membrane respectively as detection line T line and nature controlling line C line of the anti-mouse two of rabbit of H7 rabbit, concentration is 1.0 ~ 2.0mg/mL, discharge rate is 0.5 ~ 1.0 μ L/cm, 37 ℃ of vacuum drying are spent the night, sample pad, nitrocellulose membrane, thieving paper, filter paper are sticked on the PVC base plate successively, be cut into the wide strip of 4mm after posting, be installed; The test strips prepared is placed in to Fresco Bag and adds the drying agent sealing, be placed in dry cylinder and save backup; 4) utilize the double antibodies sandwich method to be measured sample; The gold-magnetic particles that captures bacterium is diluted to concentration 50 ~ 150 μ g/mL, gets 100 μ L and be added drop-wise in the test strips well, read with the immunochromatographiassays assays instrument after 10min, record the value of T line absorbance, C line absorbance and T/C; 5) qualitative analysis: the result that detects by an unaided eye is carried out qualitative analysis, the T line develops the color has Escherichia coli O 157: H7 in interpret sample, and the T line does not develop the color not to be had Escherichia coli O 157: H7 or contain Escherichia coli O 157 in interpret sample: the amount of H7 is lower than lowest detection lower limit 10
4cFU/mL; 6) quantitative test: use the immunochromatographiassays assays instrument to measure the absorbance of T line, C line, the ratio of the absorbance of T line and C line is designated as the T/C value, and the concentration of different bacterium of take is horizontal ordinate, and the T/C value is ordinate drawing standard curve; With reference to the canonical plotting of doing, determine the Escherichia coli O 157 in common sample: H7 quantity.
2. method according to claim 1, is characterized in that described step 1) nanometer gold magnetic particle particle diameter is 50nm; The amount of the nanometer gold magnetic particle of the corresponding 50nm of amount 200 ~ 300 μ g of the enterobacteria O157:H7 of step 1) Chinese People's Anti-Japanese Military and Political College monoclonal antibody is 0.5 ~ 1.0mg.
3. method according to claim 1 is characterized in that: the described immuno-chromatographic test paper strip of step 3) is to paste successively sample pad, nitrocellulose membrane, thieving paper, filter paper to form on adhesive base, there is no pad.
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