CN110261608B - Visual detection and automatic counting method for food escherichia coli colony based on magnetic fluorescent probe - Google Patents
Visual detection and automatic counting method for food escherichia coli colony based on magnetic fluorescent probe Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial detection, and relates to a visual detection and automatic counting method for food escherichia coli colonies based on a magnetic fluorescent probe; the method comprises the following specific steps: firstly, preparing a magnetic fluorescent probe, and then culturing bacterial colonies of strains in food; adding the magnetic fluorescent probe into a food strain colony plate, and putting the food strain colony plate into a portable fluorescent imager to obtain a colony fluorescent plate image; when escherichia coli exists in the bacterial colony, the specific fluorescent carbon quantum dots can be adsorbed on the bacterial colony of the escherichia coli, so that the bacterial colony of the escherichia coli shows fluorescence; otherwise, no fluorescence is displayed; therefore, the visual identification of the escherichia coli bacterial colonies is realized; then, inputting the obtained escherichia coli colony fluorescent plate image into a computer program for processing, and realizing automatic counting of escherichia coli; the method utilizes the specific fluorescent probe to position the escherichia coli colony, realizes the fluorescent visual detection of the escherichia coli colony, and can accurately calculate the content of the escherichia coli in real time.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a visual detection and automatic counting method for food escherichia coli colonies based on a magnetic fluorescent probe.
Background
The food can be polluted by the escherichia coli through different ways, and under the action of the escherichia coli, the food is rotten and deteriorated, and the due nutritional ingredients are lost, so that the edibility and the safety of the food are influenced. After people eat food polluted by escherichia coli, the food can cause parenteral infection and acute diarrhea, and diseases such as dysentery, cholera or typhoid fever and even serious septicemia are caused. It seriously harms the public health safety of human beings, and is an internationally recognized health monitoring indicator bacterium.
The existing techniques for detecting Escherichia coli mainly include conventional biochemical assay, immunoassay, and molecular biological method. The traditional biochemical determination method realizes the detection of the escherichia coli according to the growth characteristics of microorganisms, and the method is long in time consumption, easy to be influenced by the growth environment and low in accuracy. The immunoassay and molecular biology methods have the disadvantages of low reproducibility, high cost, and the like. And the above methods cannot simultaneously realize the identification and counting of Escherichia coli.
The most probable colony counting method and the plate counting method are the most commonly used escherichia coli counting methods, but the method has the disadvantages of complex operation, long time consumption and relatively high result randomness, and how to realize the quick, simple and accurate escherichia coli detection becomes a difficult point of the current research. The fluorescence sensing technology is widely concerned by researchers due to high sensitivity, low cost and strong specificity; but there has been no research applied to this field. Therefore, the research combines the magnetic fluorescent nano probe with the traditional colony plate counting method to realize the quick and accurate identification and counting of the escherichia coli.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides the escherichia coli detection method which is simple to operate and high in accuracy, and the method can shorten the detection period, simplify the detection steps, improve the detection precision and achieve the purpose of automatically and visually detecting escherichia coli.
In order to realize the purpose, the invention firstly provides a food escherichia coli colony visual detection and automatic counting method based on a magnetic fluorescent probe, which specifically comprises the following steps:
step 1, magnetic nano-microsphere Fe3O4@SiO2Preparation of cDNA: respectively adding ferric chloride hexahydrate, sodium acetate trihydrate and trisodium citrate into ethylene glycol, magnetically stirring for 8-12 hours at 40-60 ℃, transferring the mixed solution into a reaction kettle, standing for 8-16 hours at 180-200 ℃ to obtain a reacted mixed solution, washing for 3 times by deionized water and ethanol respectively, and performing vacuum drying on the magnetically separated product at 50-70 ℃ for 6-12 hours to obtain Fe3O4A nanoparticle; the using amount ratio of the ferric chloride hexahydrate, the sodium acetate trihydrate, the trisodium citrate and the glycol is 100-10 g: 200-20 g: 20-2 g: 1L;
weighing Fe3O4Dissolving nano particles in a mixed solution of ethanol and water, carrying out ultrasonic oscillation for 15-60 min to form a uniform solution, then adding ammonia water into the mixed solution, carrying out ultrasonic oscillation for 30-120 min, finally adding tetraethoxysilane into the mixed solution, carrying out ultrasonic oscillation for 45-120 min, respectively washing the obtained mixed solution for 3 times by using deionized water and ethanol, drying the magnetically separated product in a vacuum drying oven at 50-70 ℃ for 6-12 h to obtain Fe3O4@SiO2(ii) a Said Fe3O4The dosage ratio of ammonia water, ethyl orthosilicate, ethanol and water is 4-20 g: 0.05-0.2L: 0.01-0.1L: 1-5L: 1-2L;
mixing Fe3O4@SiO2Adding the mixture into toluene and aminopropyl trimethoxy silane, stirring the mixture for 20 to 30 hours at the temperature of between 100 and 150 ℃, and respectively washing the obtained mixed solution with deionized water and ethanol3 times, drying the magnetically separated product in a vacuum drying oven at 50-70 ℃ for 5-12 h to obtain Fe3O4@SiO2-NH2(ii) a Said Fe3O4@SiO2The adding amount ratio of the toluene to the aminopropyltrimethoxysilane is 10-50 g: 5-15L, 1-2L;
adding the carboxyl modified aptamer complementary strand (cDNA) into a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and fully shaking for 15-45 min; the Fe obtained is then3O4@SiO2-NH2Adding the mixture into the mixed solution and shaking for 2-8 h; the obtained product is magnetically separated and washed for a plurality of times to obtain the magnetic nano microsphere Fe3O4@SiO2-cDNA；
In Fe3O4@SiO2In cDNA, Fe3O4The particle size is 50-200 nm and SiO2The thickness is 10-50 nm; the concentration of EDC and NHS solution was 20mg/mL and the dosage ratio was 3: 1-1; the concentration range ratio of the added aptamer complementary strand is 15-35 mu mol/L; fe3O4@SiO2-NH2And the dosage ratio of the EDC/NHS solution is 0.5-0.2 mg: 1 mL; the sequence of the carboxyl modified aptamer complementary strand (cDNA) is COOH-TTAGCAAAGT AGCGTGCACT TTTG;
step 2, preparing fluorescent carbon quantum dots (aptamer-CQDs): mixing and stirring citric acid, concentrated sulfuric acid and ethylene glycol, and then placing the mixture in a microwave oven to heat for 1-5 min to obtain a Carbon Quantum Dot Solution (CQDs); centrifuging (8000-15000 rpm, 10-20 min) and dialyzing (cut-off molecular weight of a dialysis bag is 1000KD) the carbon quantum dot solution to remove impurities in the carbon quantum dot solution, so as to obtain the fluorescent carbon quantum dot with the surface rich in carboxyl; adding the EDC/NHS mixed solution into a fluorescent carbon quantum dot solution, stirring for 10-20 min, then adding an amino-modified escherichia coli aptamer (aptamer), and stirring for 2-5 h to obtain specific fluorescent carbon quantum dots (aptamer-CQDs);
the dosage ratio of the citric acid to the concentrated sulfuric acid to the ethylene glycol is 0.4-0.1 g: 0.5-0.05 mL: 1 mL; the concentration of the EDC and the NHC solution is 20mg/L, and the concentration of the EDC and the NHC solution isThe volume is (3-1) to 1; the volume ratio of the carbon quantum dot solution to the EDC/NHC solution is 1-5: 1-2; the concentration of the escherichia coli aptamer is 15-35 mu mol/L, and the sequence of the escherichia coli aptamer is GCAATGGTACGGTACTT CCCCATGAGT GTT GTGAAAT GTT GGGACACTAG GTGGCATAGAGCCGCA AAAG TGCACG CTACTTTGC TAA-NH2；
Step 3, preparing a magnetic fluorescent probe:
the Fe obtained in the step 1 is mixed3O4@SiO2-mixing and incubating cDNA and aptamer-CQDs obtained in step 2 at normal temperature, and magnetically separating to remove excessive Fe3O4@SiO2cDNA to obtain the magnetic fluorescent Probe Fe3O4@SiO2-CQDs；
Step 4, culturing bacterial colonies in food:
under aseptic condition, the food sample pretreatment method refers to national standard GB-4789.36-2016 (national food safety Standard for food microbiology inspection Escherichia coli O157: H7/NM inspection) to obtain sample dilution homogenate, and ten times of serial dilution sample homogenate with dilution gradient of 10 is prepared-(p+1)，10-(p+2)，10-(p+3)，……，10-(p+i)(p is an integer greater than or equal to zero, i is an integer greater than zero); then, carrying out coating plate culture on the sample diluted uniform solution with different gradients, inoculating n solid culture medium plates on each gradient, adding bacterial liquid into each plate for coating, then inversely placing the plates in a certain environment for culture, and obtaining food strain colony plates with different dilution gradients after culture;
and 5, rapidly identifying escherichia coli colonies:
adding the magnetic fluorescent probe in the step 3 into the food strain colony plates with different dilution gradients in the step 4, and standing for a period of time; if the colony is Escherichia coli, the Escherichia coli is combined with aptamer-CQDs, resulting in a magnetic fluorescent probe Fe3O4@SiO2Fe in CQDs3O4@SiO2-cDNA and aptamer-CQDs separation; if the colony is not Escherichia coli, Fe3O4@SiO2cDNA and aptamer-CQDs do not separate, but remain as Fe3O4@SiO2-CQDs;
the magnet is arranged above the dish cover of the bacterial colony flat plate, and separated Fe can be separated3O4@SiO2cDNA and residual Fe3O4@SiO2Separating CQDs from bacterial colonies, wherein the magnetic nano-microspheres and the magnetic fluorescent probes are adhered to the inner surface of the culture dish cover; separating the magnet and the flat dish cover from the flat dish bottom, putting the flat dish bottom into a portable fluorescence imager, and obtaining the dilution gradient of 10-(p+1)Fluorescent plate image P of n colony plates1-1、P1-2、P1-3、……、P1-n(n is an integer greater than zero); obtaining n dilution gradients of 10-(p+2)Fluorescent flat plate image P of2-1、P2-2、P2-3、……、P2-nObtaining bacterial liquid dilution gradient of 10-(p+3)Fluorescent flat plate image P of3-1、P3-2、P3-3、……、P3-n… …, obtaining a dilution gradient of 10-(p+i)Fluorescent flat plate image P ofi-1、Pi-2、Pi-3、……、Pi-n(ii) a Obtaining a total of i × n colony fluorescent plate images; at this time, specific fluorescent carbon quantum dots (aptamer-CQDs) are adsorbed on the escherichia coli bacterial colony, so that the escherichia coli bacterial colony shows fluorescence; meanwhile, aptamer-CQDs cannot be adsorbed on non-Escherichia coli colonies, and the non-Escherichia coli colonies do not show fluorescence; therefore, the quick and visual identification of the escherichia coli bacterial colony is realized;
step 6, automatically counting escherichia coli colonies in food:
(1) preprocessing the i × n colony fluorescent plate images acquired in the step 5, and performing linear filtering on the colony fluorescent plate images by using a color image filtering function imfilter to remove noise in the images;
(2) threshold segmentation: extracting a blue (B) channel component image of the preprocessed image, and graying the component image to obtain a threshold value T of the B component image; segmenting the escherichia coli bacterial colony by using a threshold T, setting the pixel point higher than the threshold T as 1, and setting the pixel point lower than the threshold T as 0, so as to obtain a fluorescent plate bacterial colony segmentation image;
(3) fluorescence plate colony counting: detecting colony edges of the fluorescence plate colony segmentation images by adopting an eight-neighborhood edge tracking method, and counting escherichia coli colonies by utilizing a connected region counting method; counting colonies of the obtained i × n fluorescence plate colony segmentation images (i and n are integers larger than zero, i dilution gradients are n times, each gradient is repeated), calculating average colony number of Escherichia coli of each dilution gradient, and the dilution gradient is 10-(p+1)The average number of colonies of Escherichia coli was designated as N1Dilution gradient of 10-(p+2)The average number of colonies of Escherichia coli was designated as N2Dilution gradient of 10-(p+3)The average number of colonies of Escherichia coli is N3… … dilution gradient of 10-(p+i)The average number of colonies of Escherichia coli is NiWherein p is an integer greater than or equal to zero, i and n are integers greater than zero, and the total number of Escherichia coli colonies in the food is further automatically calculated according to a calculation formula.
The calculation formula is specifically divided into 4 types:
A. if there is only one dilution gradient (10)-(p+k)) And counting the number of escherichia coli colonies on the dilution gradient colony plate when the number of escherichia coli colonies on the plate is between 20 and 200CFU, wherein the number of the escherichia coli colonies in the sample is C (CFU/mL) ═ Nk/(V×10-(p+k)) (C is the number of E.coli colonies in the food sample, NkThe number of Escherichia coli colonies, 10-(p+k)K is an integer greater than or equal to zero and less than i) as a dilution gradient value.
B. If the number of E.coli colonies on all dilution gradient colony plates is less than 20CFU, N1，N2，N3，……，NiIf the concentration is less than 20CFU, counting the number of coliform colonies on the colony plate with the lowest dilution gradient, wherein the number of colonies in the sample is C (CFU/mL) ═ N1/(V×10-(p+1)) (C is the number of E.coli colonies in the food sample, N1Number of E.coli colonies at minimum dilution gradient, 10-(p+1)The lowest dilution gradient value).
C. If a certain dilution ladderDegree (10)-(p+k)) The colony number of the colony plate is more than 200CFU, but no escherichia coli colony exists on the next gradient plate, or although the escherichia coli colony exists, but the colony number is not in the range of 20-200 CFU, the escherichia coli colony on the dilution gradient culture dish is counted, and the colony number in the food sample is C (CFU/mL) ═ Nk/(V×10-(p+k)) (C is the number of E.coli colonies in the food sample, NkThe number of Escherichia coli colonies, 10-(p+k)K is an integer greater than or equal to zero and less than i) as a dilution gradient value.
D. If there are 2 continuous gradients (10)-(p+k)And 10-(p+k+1)) The colony number of the colony plate is between 20 and 200CFU, and the colony number in the food sample is C (CFU/mL) ═ Nk+Nk+1)/(1.1V×10-(p+k)) (C is the number of E.coli colonies in the food sample, NkFor low dilution gradient E.coli colony number, Nk+1The number of Escherichia coli colonies with high dilution gradient is 1.1, a calculation coefficient is 10-(p+k)A dilution gradient value for a low dilution gradient, k being an integer greater than or equal to zero and less than i).
Preferably, the incubation time in the step 3 is 12-24 h.
Preferably, Fe in step 33O4@SiO2The volume ratio of the cDNA to the aptamer-CQDs is (2-1): 1.
preferably, the volume of the added bacteria liquid for each plate in the step 4 is 0.1 mL-1 mL.
Preferably, the temperature of the culture in the step 4 is 35-37 ℃, and the time is 10-15 h.
Preferably, the dosage of the magnetic fluorescent probe in the step 5 is 1-5 mL.
Preferably, the standing time in the step 5 is 10-20 min.
The invention has the beneficial technical effects that:
(1) compared with single magnetic nano material or fluorescent material, the invention prepares the specific magnetic fluorescent probe (Fe)3O4@SiO2CQDs), the fluorescent probe is composed of magnetic nanospheres Fe3O4@SiO2And fluorescent carbon quantum dots are compounded; in which Fe3O4@SiO2Overcome Fe3O4The magnetic nano particles are easy to agglomerate and settle, the biocompatibility is poor and the like, and the water solubility and the stability of the probe are improved. The carbon quantum dots have good biocompatibility and can be well combined with escherichia coli colonies, the magnetic fluorescent probe can accurately identify the escherichia coli colonies in a mixed strain flat plate, and the magnetic fluorescent probe is attached to the surface of the escherichia coli colonies to display fluorescence, so that the escherichia coli colonies can be identified.
(2) Compared with the most probable number counting method of escherichia coli in the national standard, the method is based on the fluorescence plate counting method, and the obtained escherichia coli colony fluorescence plate image is processed to realize automatic counting of the escherichia coli, so that the content of the escherichia coli in the food sample can be tracked and known in real time, intuitively and accurately.
(3) Compared with a colibacillus flat plate counting method in the national standard, the method only uses the most common and most common solid culture medium, and utilizes a specific fluorescent probe to position the colibacillus colony, thereby realizing the fluorescent visual detection of the colibacillus colony; the automation and the intellectualization of the colony counting are realized by utilizing the image processing technology. In addition, the method can accurately identify the escherichia coli bacterial colony from a large number of unknown bacterial colony strains, can avoid the false positive phenomenon caused by using some identifying culture media, achieves the aim of identifying the escherichia coli more accurately and quickly, and realizes the aim of automatically and visually detecting the escherichia coli.
Drawings
FIG. 1(a) is a TEM image of magnetic nanospheres; (b) the fluorescence spectrum curve of the magnetic fluorescent probe is shown in the figure, and the fluorescent picture of the magnetic fluorescent probe under 365nm exciting light is shown in the figure.
FIG. 2 is an image of a colony plate under natural light, and the dilution gradients in FIGS. (a), (b), (c), (d) and (e) are 10-1，10-2，10-3，10-4，10-5。
FIG. 3 is an image of a colony fluorescent plate obtained after addition of a magnetic fluorescent probe, and the dilution gradients of FIGS. (a), (b), (c), (d) and (e) are 10-1，10-2，10-3，10-4，10-5。
In FIG. 4, (a), (b), (c), (d) and (e) are each a dilution gradient of 10-1，10-2，10-3，10-4And 10-5The colony segmentation image of the fluorescence plate of (1).
Detailed description of the preferred embodiment
The present invention will be described in further detail with reference to the following detailed description of the drawings, but the scope of the present invention is not limited thereto.
Example 1:
firstly, culturing strains in milk in a solid state to obtain a strain colony plate; then adding the prepared specific magnetic fluorescent probe into a colony flat plate, and magnetically separating out the magnetic fluorescent probe which does not react with escherichia coli; then, acquiring an escherichia coli colony fluorescent plate image; and finally, carrying out image processing to realize automatic counting of escherichia coli colonies.
Step 1, magnetic nano-microsphere Fe3O4@SiO2Preparation of cDNA:
sequentially adding 1.08g of ferric chloride hexahydrate, 1.8g of sodium acetate trihydrate and 0.25g of trisodium citrate into 50mL of ethylene glycol, magnetically stirring for 9 hours at 40 ℃ to form a uniform and transparent solution, transferring the solution into a reaction kettle, standing for 10 hours at 200 ℃, finally washing the obtained mixed solution for 3 times by using deionized water and ethanol respectively, magnetically separating to obtain a black product, and performing vacuum drying for 10 hours at 60 ℃ to obtain Fe3O4A nanoparticle powder. 0.1g of Fe was weighed3O4Adding the mixture into a mixed solution of ethanol (40mL) and water (10mL), carrying out ultrasonic oscillation for 30min, then adding 1.2mL of ammonia water into the mixed solution, carrying out ultrasonic oscillation for 60min, finally adding 0.35mL of ethyl orthosilicate into the mixed solution, carrying out ultrasonic oscillation for 60min, respectively washing the obtained mixed solution for 3 times by using deionized water and ethanol, carrying out magnetic separation to obtain a black product, and carrying out vacuum drying for 8h at 60 ℃ to obtain Fe3O4@SiO2。
0.1g of Fe3O4@SiO2Added to toluene (50mL) and aminopropyltrimethoxysilaneStirring the mixed solution of alkane (5mL) for 24h at 120 ℃, washing the obtained mixed solution for 3 times by using deionized water and ethanol respectively, performing magnetic separation to obtain a black product, and drying the black product in a vacuum drying oven at 60 ℃ for 8h to obtain Fe3O4@SiO2-NH2. Adding the carboxyl modified aptamer complementary strand into EDC/NHS mixed solution (9mL), and shaking for 25min, wherein the concentration of the aptamer complementary strand is 20 mu M; the Fe obtained is then3O4@SiO2-NH2Adding into the mixture and shaking for 4 h. The obtained product is magnetically separated and washed for a plurality of times to obtain the nano-microsphere Fe3O4@SiO2-a cDNA solution. The base sequence of the adapter complementary strand was COOH-TTAGCAAAGT AGCGTGCACT TTTG.
Fe can be seen by transmission electron microscopy (FIG. 1a)3O4The average diameter of the nanoparticles is 95nm, the average thickness of the silica shell is 40nm, and Fe3O4@SiO2-NH2The cDNA has an obvious nucleocapsid structure. The property of the magnetic nano microsphere is more single magnetic nano particle Fe3O4Is stable because the silica shell can block Fe3O4The nano particles are agglomerated and can protect Fe3O4The nanoparticles are not destroyed in harsh environments.
Step 2, preparing fluorescent carbon quantum dot aptamer-CQDs:
stirring and mixing 2g of citric acid, 100 mu L of concentrated sulfuric acid and 10mL of polyethylene glycol 200, placing the mixture in a microwave oven for heating for 2min, centrifuging the obtained product (10000rpm, 15min) to remove insoluble particles, and dialyzing (the molecular weight of a dialysis bag is 1000KD) to remove unreacted micromolecules, thereby obtaining the carbon quantum dots with surfaces rich in carboxyl. Adding the carbon quantum dot solution (1mL) into an EDC/NHS mixed solution (9mL) and stirring for 15min, then adding the amino-modified aptamer and stirring for 3h, wherein the final concentration of the aptamer is 20 mu M, and finally obtaining the aptamer-modified carbon quantum dot solution (aptamer-CQDs). The sequence of the escherichia coli aptamer is GCAATGGTACGGTACTT CCCCATGAGT GTT GTGAAATGTT GGGACACTAG GTGGCATAGAGCCGCA AAAG TGCACG CTACTTTGCTAA-NH2。
Step 3, preparing a magnetic fluorescent probe:
1.2mL of Fe obtained in step 13O4@SiO2Incubating the mixture of cDNA and 1mL of aptamer-CQDs obtained in step 2 for 15h, and removing excessive aptamer-CQDs by magnetic separation to obtain a high-magnetism fluorescent probe Fe3O4@SiO2CQDs. As can be seen from FIG. 1b, the magnetic fluorescent probe has excellent fluorescent characteristics, the optimal emission wavelength is 450nm, and blue fluorescence is displayed under the excitation of 365nm excitation light.
Step 4, culturing bacterial colonies in milk:
the pretreatment steps of filtering and diluting the milk sample are carried out according to GB4789.36-2016 test for Escherichia coli O157: H7/NM in national food safety Standard food microbiology. Under aseptic condition, putting 25mL milk into an aseptic conical flask containing 225mL normal saline, then putting the sample solution into a high-speed dispersion machine for dispersion, fully and uniformly mixing, filtering the centrifuged sample filtrate, sequentially preparing the sample filtrate into ten-fold-increasing serial diluted sample uniform solutions, and obtaining sample dilution gradients of 10-1，10-2，10-3，10-4And 10-5. Inoculating 1mL of sample homogenate to 1 common solid culture medium in each gradient, spreading uniformly, culturing at 36 + -1 deg.C for 12h to obtain 5 colony plates of milk strain, and acquiring colony plate image under natural light (figure 2), wherein (a), (b), (c), (d) and (e) in figure 2 are respectively inoculated with dilution gradient of 10-1，10-2，10-3，10-4And 10-5And (4) a colony plate image of the sample bacterium liquid. As can be seen from FIG. 2, the E.coli colonies were similar to those of other strains and could not be identified.
And 5, rapidly identifying escherichia coli colonies:
2mL of a magnetic fluorescent probe was added to the colony plate in step 4, and left to stand for 15 min. If the colonies are E.coli, the colonies will bind to aptamer-CQDs, resulting in Fe3O4@SiO2And aptamer-CQDs; if the colonies are not E.coli colonies, Fe3O4@SiO2And aptamer-CQDs do not separate, but are still Fe3O4@SiO2The form CQDs exists. The magnet is arranged above the culture dish cover, and the separated magnetic nano material Fe can be separated3O4@SiO2cDNA and residual Fe3O4@SiO2CQDs are detached from the colony plates and attached to the inner surface of the culture dish lid. After the magnet and the flat dish cover are separated from the flat dish bottom, the flat dish bottom is placed into a portable fluorescence imager to obtain a colony fluorescence flat image (shown in figure 3). In FIG. 3, (a), (b), (c), (d) and (e) were each inoculated with a dilution gradient of 10-1，10-2，10-3，10-4And 10-5And (3) a colony fluorescent plate image of the sample bacterial liquid. As can be seen from FIG. 3, fluorescent carbon quantum dots (aptamer-CQDs) remain on the Escherichia coli colonies, so that the Escherichia coli colonies show blue fluorescence; while aptamer-CQDs did not remain on non-E.coli colonies, other colonies showed no fluorescence. Thus, the identification of E.coli colonies was achieved.
Step 6, counting the Escherichia coli colony fluorescent plates in milk:
the Escherichia coli colonies in the fluorescent plate are automatically counted through an image processing tool, and the counting steps in the software mainly comprise:
(1) preprocessing of the image: filtering the 5 fluorescent flat images in the step 5 by utilizing a color image filtering function imfilter to remove noise in the images;
(2) threshold segmentation: and extracting a blue (B) channel component image of the preprocessed fluorescent flat plate image, graying the color image of the component image, and selecting a segmentation threshold value of the fluorescent flat plate image to be 0.3. The escherichia coli colony is segmented by using a threshold value of 0.3, the pixel point higher than the threshold value is determined as 1, and the pixel point lower than the threshold value is determined as 0, so that a fluorescence plate colony segmentation image is obtained (shown in figure 4). FIGS. 4(a), (b), (c), (d) and (e) are each a dilution gradient of 10-1，10-2，10-3，10-4And 10-5The colony segmentation image of the fluorescence plate;
(3) and (3) counting colonies: and (3) counting the colonies of the 5 fluorescence plate colony segmentation images obtained in the step (6) and (2), detecting the colony edges by adopting an eight-neighborhood edge tracking method and counting the escherichia coli colonies by utilizing a connected region counting method. The fluorescent plate counting step of the invention firstly obtains a fluorescent plate image by using a fluorescent imaging technology, inputs the fluorescent plate image into a computer, then carries out image preprocessing, image segmentation and colony counting in the image on the fluorescent plate image by using computer automatic counting software, and calculates the average colony number of escherichia coli of each gradient to obtain the following results:
TABLE 1 count of colony number in plates for each dilution
(4) Fluorescence plate counting of Escherichia coli in milk
Coli counts in fluorescent plates were in accordance with the fourth case, i.e. the E.coli colonies in 2 plates with continuous gradients were all between 20-200 CFU, according to the formula C (CFU/mL) ═ N (N)k+Nk+1)/(1.1V×10-(p+k)) Calculation (C is the number of E.coli colonies in the milk sample, NkTotal number of E.coli colonies at low dilution, Nk+1The total number of Escherichia coli colonies at high dilution is 1.1, calculation coefficient is 10-(p+k)Dilution at low dilution), the number of E.coli in milk is 7X 103CFU/mL. The colony count obtained by the Escherichia coli plate counting method in the national standard method is 6 multiplied by 103CFU/mL. The results show that the method of the invention is consistent with the standard method, indicating the reliability of the invention.
The plate counting method in the national standard method is to select typical and suspicious colonies, select a part of suspected escherichia coli colonies in a broth tube and observe the gas production condition. If the broth tube produces gas, the colony can be reported as Escherichia coli positive, and the Escherichia coli number is obtained according to the number of positive tubes in the Escherichia coli broth tube. Because the traditional escherichia coli plate counting method has high randomness and is greatly influenced by the environment, the colony counting result is generally low, and the escherichia coli is required to be cultured again to confirm whether the escherichia coli is detected. Compared with the traditional method, the method provided by the invention is simple to operate, does not need to verify the test, and can be used for rapidly, accurately and automatically detecting the number of escherichia coli.
FIG. 1(a) is a TEM image of magnetic nanospheres; (b) the graph is a fluorescence spectrum curve of the magnetic fluorescent probe, and the inset is a fluorescence picture of the magnetic fluorescent probe under the excitation of 365nm exciting light. It can be seen from the figure that the magnetic fluorescent nanoprobe is successfully prepared by the invention, and the magnetic fluorescent probe shows strong blue fluorescence.
FIG. 2 is an image of a colony plate under natural light. It can be seen from the figure that the plate is a mixture of bacterial colonies of various strains in food, and whether the bacterial colonies are escherichia coli bacterial colonies or not can not be judged by naked eyes, so that the aim of counting escherichia coli can not be fulfilled.
FIG. 3 is a colony fluorescent plate image obtained after addition of a magnetic fluorescent probe; coli colony shows blue fluorescence in this flat board, and non-escherichia coli colony then does not show fluorescence, shows that magnetism fluorescence probe can the specificity identification escherichia coli colony, utilizes naked eye can distinguish the escherichia coli colony from the mixed colony flat board.
FIG. 4 is a fluorescent plate colony segmentation image. White region is the escherichia coli bacterial colony in this image, and black is the background, utilizes the characteristics of fluorescence flat image can be fast and accurately cut apart out the escherichia coli bacterial colony, realizes the purpose that the escherichia coli is quick, accurate to count.
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (7)
1. A visual detection and automatic counting method for food escherichia coli colonies based on a magnetic fluorescent probe is characterized by comprising the following steps:
step 1, preparation of magnetic nano-microspheres, marked as Fe3O4@SiO2-cDNA；
Respectively adding ferric chloride hexahydrate, sodium acetate trihydrate and trisodium citrate into ethylene glycol, magnetically stirring for 8-12 hours at 40-60 ℃, transferring the mixed solution into a reaction kettle, standing for 8-16 hours at 180-200 ℃ to obtain a reacted mixed solution, washing for 3 times by deionized water and ethanol respectively, and performing vacuum drying on the magnetically separated product at 50-70 ℃ for 6-12 hours to obtain Fe3O4A nanoparticle; the using amount ratio of the ferric chloride hexahydrate, the sodium acetate trihydrate, the trisodium citrate and the glycol is 10-100 g: 20-200 g: 2-20 g: 1L;
weighing Fe3O4Dissolving nanoparticles in a mixed solution of ethanol A and water, carrying out ultrasonic oscillation for 15-60 min to form a uniform solution, then adding ammonia water into the mixed solution, carrying out ultrasonic oscillation for 30-120 min, finally adding tetraethoxysilane into the mixed solution, carrying out ultrasonic oscillation for 45-120 min, washing the obtained mixed solution 3 times respectively by using deionized water and ethanol B, drying the magnetically separated product in a vacuum drying oven at 50-70 ℃ for 6-12 h to obtain Fe3O4@SiO2(ii) a Said Fe3O4The dosage ratio of ammonia water, ethyl orthosilicate, ethanol A and water is 4-20 g: 0.05-0.2L: 0.01-0.1L: 1-5L: 1-2L;
mixing Fe3O4@SiO2Adding the mixture into toluene and aminopropyltrimethoxysilane, stirring for 20-30 h at 100-150 ℃, washing the obtained mixed solution for 3 times by using deionized water and ethanol respectively, and drying the magnetically separated product in a vacuum drying oven at 50-70 ℃ for 5-12 h to obtain Fe3O4@SiO2-NH2(ii) a Said Fe3O4@SiO2The adding amount ratio of the toluene to the aminopropyltrimethoxysilane is 10-50 g: 5-15L, 1-2L;
adding the carboxyl modified aptamer complementary chain into a mixed solution of EDC and NHS, recording as an EDC/NHS solution, and fully shaking for 15-45 min; the Fe obtained is then3O4@SiO2-NH2Adding EDC/NHS solutionOscillating for 2-8 h; the obtained product is magnetically separated and washed for a plurality of times to obtain the magnetic nano microsphere Fe3O4@SiO2-cDNA；
In Fe3O4@SiO2In cDNA, Fe3O4The particle size is 50-200 nm and SiO2The thickness is 10-50 nm; the concentration of the EDC and the concentration of the NHS solution are both 20mg/mL, and the dosage ratio is 3: 1; the concentration range of the carboxyl modified aptamer complementary strand is 15-35 mu mol/L; said Fe3O4@SiO2-NH2And the dosage ratio of the EDC/NHS solution is 0.5-0.2 mg: 1 mL; the complementary strand of the carboxyl modified aptamer, namely the sequence of the cDNA is COOH-TTAGCAAAGT AGCGTGCACT TTTG;
step 2, preparing fluorescent carbon quantum dots, and marking as aptamer-CQDs;
mixing and stirring citric acid, concentrated sulfuric acid and ethylene glycol, and then placing the mixture in a microwave oven to heat for 1-5 min to obtain a carbon quantum dot solution; centrifuging and dialyzing the carbon quantum dot solution to remove impurities in the carbon quantum dot solution to obtain the fluorescent carbon quantum dot with the surface rich in carboxyl; adding the EDC/NHS mixed solution into the fluorescent carbon quantum dot solution, stirring for 10-20 min, then adding the amino-modified escherichia coli aptamer, and stirring for 2-5 h to obtain specific fluorescent carbon quantum dots, namely aptamer-CQDs;
the dosage ratio of the citric acid to the concentrated sulfuric acid to the ethylene glycol is 0.4-0.1 g: 0.5-0.05 mL: 1 mL;
the centrifugation condition is 8000-15000 rpm for 10-20 min; the cut-off molecular weight of the dialysis bag in the dialysis process is 1000 KD;
the concentration of the EDC solution and the NHC solution is 20mg/L, and the volume of the EDC solution and the volume of the NHC solution are (1-3): 1; the volume ratio of the carbon quantum dot solution to the EDC/NHC solution is 1-5: 1-2;
the final concentration of the escherichia coli aptamer is 15-35 mu mol/L, and the sequence of the escherichia coli aptamer is GCAATGGTACGGTACTT CCCCATGAGT GTT GTGAAAT GTT GGGACACTAG GTGGCATAGAGCCGCA AAAG TGCACG CTACTTTGC TAA-NH2；
Step 3, magnetic fluorescent probe Fe3O4@SiO2Of CQDsPreparation, Fe obtained in step 13O4@SiO2-mixing and incubating cDNA and aptamer-CQDs obtained in step 2 at normal temperature, and magnetically separating to remove excessive Fe3O4@SiO2cDNA to obtain the magnetic fluorescent Probe Fe3O4@SiO2-CQDs；
Step 4, culturing bacterial colonies of the strains in the food; diluting food sample, preparing ten times of dilution sample solutions with dilution gradient of 10-(p+1)，10-(p+2)，10-(p+3)，……，10-(p+i)Wherein p is an integer greater than or equal to zero and i is an integer greater than zero; then, performing coating plate culture on the sample diluted uniform solution with different gradients, and inoculating n solid culture medium plates to each gradient, wherein n is an integer greater than zero; adding bacterial liquid into each flat plate for coating, then inverting the flat plates in a certain environment for culturing, and obtaining food strain colony flat plates with different dilution gradients after culturing;
step 5, quickly identifying the escherichia coli bacterial colony; the magnetic fluorescent probe Fe prepared in the step 33O4@SiO2-CQDs are added to the food strain colony plates of different dilution gradients in step 4 and left for a period of time; if the colony is Escherichia coli, the Escherichia coli binds to aptamer-CQDs, resulting in Fe3O4@SiO2-cDNA and aptamer-CQDs separation; if the colony is not Escherichia coli, Fe3O4@SiO2-cDNA and aptamer-CQDs do not separate, as Fe3O4@SiO2-CQDs;
adsorbing Fe in colony plate by using magnet3O4@SiO2-cDNA and Fe3O4@SiO2Separating CQDs from bacterial colonies, placing the bottom of the plate dish with bacterial colonies in a portable fluorescence imaging instrument, and obtaining a dilution gradient of 10-(p+1)The fluorescence plate image of the n colony plates of (1) is marked as P1-1、P1-2、P1-3、……、P1-n N is an integer greater than zero; obtaining n dilution gradients of 10-(p+2)Fluorescent flat plate image P of2-1、P2-2、P2-3、……、P2-nObtaining bacterial liquid dilution gradient of 10-(p+3)Fluorescent flat plate image P of3-1、P3-2、P3-3、……、P3-n… …, obtaining a dilution gradient of 10-(p+i)Fluorescent flat plate image P ofi-1、Pi-2、Pi-3、……、Pi-nObtaining i × n colony fluorescent plate images in total, wherein i and n are integers larger than zero;
if the colony contains Escherichia coli, aptamer-CQDs can be adsorbed on the Escherichia coli colony, and the Escherichia coli colony shows fluorescence; if no Escherichia coli aptamer-CQDs exist in the colony, the colony can not be adsorbed on a non-Escherichia coli colony, and the non-Escherichia coli colony does not show fluorescence; therefore, the quick and visual identification of the escherichia coli bacterial colony is realized;
step 6, automatically counting the escherichia coli colonies in the food;
(1) preprocessing the i × n colony fluorescent plate images acquired in the step 5, and performing linear filtering on the colony fluorescent plate images by using a color image filtering function imfilter to remove noise in the images;
(2) threshold segmentation: extracting a blue (B) channel component image of the preprocessed image, and graying the component image to obtain a threshold value T of the B component image; segmenting the escherichia coli bacterial colony by using a threshold T, setting the pixel point higher than the threshold T as 1, and setting the pixel point lower than the threshold T as 0, so as to obtain a fluorescent plate bacterial colony segmentation image;
(3) fluorescence plate colony counting: detecting colony edges of the fluorescence plate colony segmentation images by adopting an eight-neighborhood edge tracking method, and counting escherichia coli colonies by utilizing a connected region counting method; counting colonies of the obtained i × n fluorescence plate colony segmentation images, and calculating the average colony number of Escherichia coli in each dilution gradient of 10-(p+1)The average number of colonies of Escherichia coli was designated as N1Dilution gradient of 10-(p+2)The average number of colonies of Escherichia coli was designated as N2Dilution gradient of 10-(p+3)The average number of colonies of Escherichia coli is N3… …, diluteRelease gradient of 10-(p+i)The average number of colonies of Escherichia coli is NiWherein p is an integer greater than or equal to zero, i and n are integers greater than zero, and the total number of escherichia coli colonies in the food is automatically calculated according to a calculation formula;
the calculation formula is specifically divided into 4 types:
A. if there is only one dilution gradient (10)-(p+k)) And counting the number of escherichia coli colonies on the dilution gradient colony plate if the number of escherichia coli colonies on the plate is between 20 and 200CFU, wherein the calculation formula of the number of the colonies in the sample is C (CFU/mL) = Nk /(V×10-(p+k)) (ii) a Wherein C is the number of Escherichia coli colonies in the food sample, and NkThe number of Escherichia coli colonies, 10-(p+k)The value is a dilution gradient value, k is an integer smaller than i, i is an integer larger than zero, and V is the volume of the food sample bacterium liquid added when the flat plate is coated, and the unit is mL;
B. if the number of E.coli colonies on all dilution gradient colony plates is less than 20CFU, N1，N2，N3，……，NiIf the number of the Escherichia coli colonies on the colony plate with the lowest dilution gradient is less than 20CFU, counting the number of the Escherichia coli colonies on the colony plate with the lowest dilution gradient, wherein the calculation formula of the number of the Escherichia coli colonies in the sample is C (CFU/mL) = N1 /(V×10-(p+1)) (ii) a Wherein C is the number of Escherichia coli colonies in the sample, and N is1Number of E.coli colonies at minimum dilution gradient, 10-(p+1)The value is the lowest dilution gradient value, V is the volume of the food sample bacteria liquid added when the flat plate is coated, and the unit is mL;
C. if a certain dilution gradient (10)-(p+k)) The colony number of the colony plate is more than 200CFU, but no escherichia coli colony exists on the next gradient plate, or although the escherichia coli colony exists, but the colony number is not in the range of 20-200 CFU, the escherichia coli colony on the dilution gradient culture dish is counted, and the calculation formula of the colony number in the food sample is C (CFU/mL) = Nk /(V×10-(p+k)) (ii) a Wherein C is the number of Escherichia coli colonies in the food sample, and NkThe number of Escherichia coli colonies, 10-(p+k)Is a dilution gradient value, k is an integer greater than or equal to zero and less than i, and V is the volume of the sample bacteria liquid added during the plate coatingIn units of mL;
D. if there are 2 continuous gradients (10)-(p+k)And 10-(p+k+1)) The colony number of the colony plate is between 20 and 200CFU, and the calculation formula of the colony number in the food sample is C (CFU/mL) = (N)k+Nk+1) /(1.1V×10-(p+k)) (ii) a Wherein C is the number of Escherichia coli colonies in the food sample, and NkFor low dilution gradient E.coli colony number, Nk+1The number of Escherichia coli colonies with high dilution gradient is 1.1, a calculation coefficient is 10-(p+k)The dilution gradient value of the low dilution gradient is shown, k is an integer which is more than or equal to zero and less than i, and V is the volume of the food sample bacterium liquid added when the flat plate is coated, and the unit is mL.
2. The visual detection and automatic counting method for food escherichia coli colonies based on the magnetic fluorescent probe as claimed in claim 1, wherein the incubation time in step 3 is 12-24 hours.
3. The visual detection and automatic counting method for food escherichia coli colonies based on the magnetic fluorescent probe as claimed in claim 1, wherein the Fe is obtained in step 33O4@SiO2The volume ratio of the cDNA to the aptamer-CQDs is (2-1): 1.
4. the food escherichia coli colony visual detection and automatic counting method based on the magnetic fluorescent probe as claimed in claim 1, wherein the volume of the added bacterial liquid for each plate in the step 4 is 0.1-1 mL.
5. The visual detection and automatic counting method for the food escherichia coli colonies based on the magnetic fluorescent probe as claimed in claim 1, wherein the temperature for the culture in the step 4 is 35-37 ℃ and the time is 10-15 hours.
6. The magnetic fluorescent probe-based food escherichia coli colony visual detection and automatic counting method as claimed in claim 1, wherein the amount of the magnetic fluorescent probe used in step 5 is 1-5 mL.
7. The visual detection and automatic counting method for food escherichia coli colonies based on the magnetic fluorescent probe as claimed in claim 1, wherein the standing time in step 5 is 10-20 min.
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