CN105021585A - Method for detecting food-borne pathogenic bacteria on basis of metal organic framework material and aptamer fluorescence sensor - Google Patents
Method for detecting food-borne pathogenic bacteria on basis of metal organic framework material and aptamer fluorescence sensor Download PDFInfo
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- CN105021585A CN105021585A CN201510479496.XA CN201510479496A CN105021585A CN 105021585 A CN105021585 A CN 105021585A CN 201510479496 A CN201510479496 A CN 201510479496A CN 105021585 A CN105021585 A CN 105021585A
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Abstract
The invention provides a method for detecting food-borne pathogenic bacteria on the basis of a metal organic framework material and aptamer fluorescence sensor. The fluorescence quenching characteristic of a metal organic framework material and the adsorbability of the framework material to aptamer are utilized, when the aptamer marked by a fluorescence probe is adsorbed to the metal organic framework material, fluorescence of the probe is quenched, a target bacterium is added into a system, the aptamer marked by the fluorescence probe leaves the metal organic framework material and is combined with the target bacterium to enhance fluorescence signals of the probe, and by means of the combination with high appetency and a high specific recognition ability of the aptamer, the method is constructed. Salmonellas are used as model analyte, the fluorescence intensity of the probe and the logarithm of target bacterium concentration form a good linear relation, the linearity range is 18-3.2*10<4> cfu/mL, the detection limit is 5 cfu/mL (S/N=3), the relative standard deviation (RSD) of standard adding experiments ranges from 3.6% to 7.5%, and the recovery rate is within the range of 90.0%-106.0%. The method for detecting the food-borne pathogenic bacteria has the advantages of being accurate, sensitive, high in specificity and the like.
Description
Technical field
The present invention relates to food-borne pathogens and analyze detection field, particularly relate to the method utilizing metal-organic framework materials fluorescent quenching characteristic and aptamer recognition technology to detect food-borne pathogens.
Background technology
Food-borne pathogens is that carrier causes one of human diseases large bacterioid with food, have source various, breed feature rapidly, be the primary food safety hazard factor.
Current, in food, pathogenic bacteria commonly use detection technique general culture method, immunization, polymerase chain reaction method (PCR), gene probe method etc.(1) general culture method is based upon microorganism increasing bacterium, is separated and cultivates and (Crit Rev Microbiol on biochemical identification basis, 2011,37 (1): 40), cannot detect difficulty cultivation or not educable pathogenic bacteria, and specificity is not high, sensitivity is low, operates loaded down with trivial details consuming time, can not realize effectively monitoring prevention effect.(2) immunization is based on combination (Biosens Bioelectron specific between antigen and corresponding antibodies, 2000,15 (11-l2): 549), between different bacterium, may common antigen be there is between bacterium and host, therefore only can not judge malignant bacteria according to antigen detection, in addition, immunization false positive probability is higher, detection influence factor is more, limits the widespread use of this method.(3) PCR is a species specific DNA cloning technology (J Microbiol Methods, 2007,69 (1): 1), there is the advantages such as detection speed is fast, efficiency is high, easy to use, but also there is many problems in actual detection, the kind as detected pathogenic microorganisms is less, and resolution is low, may there is interference between primer, there is certain false positive and false negative phenomenon in testing result.(4) gene probe method is also known as making nucleic acid molecular hybridization method (Emerg Infect Dis, 2011,17 (1): 7; J Dairy Sci, 2009,92 (7): 3027), there is high specific and susceptibility, the qualification of microorganism can be directly used in.Its principle oneself is known nucleotide sequence DNA fragmentation isotope or additive method mark, add in the tested DNA sample of own sex change, under certain condition can with this sample in have the region of DNA section of homologous sequence to form heteroduplex, thus reach the object of DNA in qualification sample.The method is comparatively extensive in field of detection of food safety application, can sensitive, quick, directly, stably detect pathogenic microorganisms, and not by the impact of non-pathogenic microorganism, but also have some limitations, comprise be difficult to be separated from the food product environment of complexity, the DNA of enrichment and purifying high quality, need different hybridization conditions etc. when detecting various pathogens, these all easily cause non-specific hybridization, thus cause false positive or false negative result.In addition, carry out DNA hybridization and generally need 10
4~ 10
5the copy of individual pathogenic bacteria target gene, therefore still needs to carry out Zengjing Granule, which has limited the development of gene probe as a kind of Fast Detection Technique.Separately there is patented technology (Chinese patent, application number: 201410070122.8) exempt from mark aptamer sensor based on dyestuff AccuBlue and detect food-borne pathogens, devise " signal on " and " signal off " two kinds of detecting patterns, committed step is still making nucleic acid molecular hybridization, very limited when detecting actual complex sample.
Metal-organic framework materials is the novel porous materials that a class has huge applications prospect, is to be assembled by molecule by transition metal ion or metal cluster and organic ligand and the crystalline compounds with infinite network structure that crystal engineering method builds.Under extraneous physics, the effect of chemical pumping thing (as photon, electronics, ion, molecule etc.), metal-organic framework materials can produce different responses, as the change etc. of energy trasfer, electro transfer, framework conformation and crystal formation, be applied in gas storage, Selective Separation, heterocatalysis, magnetics and the aspect such as optics, drug delivery more, research in molecule sensing is in the ascendant, the aspects such as kation, negative ion, Small molecular and gas detect are applied in, such as, in aqueous solution, Eu
2(FMA)
2(OX) (H
2o)
44H
2o material is to Cu
2+have very high luminous sensitivity and selectivity (Chem Commun, 2010,46:5503), Zn-Ir (2-phenylpyridine) material alternative detects O
2(J Am Chem Soc, 2010,132:922), has good stability and reappearance.
Part in metal-organic framework materials, usually containing conjugated pi electron system and a large amount of holes, allows nucleic acid molecules combine with it and reconstruct; In addition, as the metallic ion of tie point as Zn
2+and Cu
2+deng, there is inherent Fluorescence Quenching Characteristics; The character of part and metallic ion metal-organic framework materials is had concurrently adsorbs nucleic acid molecule and quenching fluorescence ability.The research of new structure metallic organic framework porosint and the application in biomolecule and Food Safety Analysis thereof need to be furtherd investigate further.
Summary of the invention
The technical problem to be solved in the present invention is: overcome existing pathogenic microbes detect technology specificity not high, sensitivity is low, operate loaded down with trivial details consuming time, the high deficiency of false positive rate, metal-organic framework materials is applied to Food Safety Analysis field, utilize the fluorescent quenching characteristic of metal-organic framework materials and the adsorbability to aptamer, the high-affinity that bind nucleic acid is fit and high specific recognition capability, provide a kind of fast, accurately and reliably, the food-borne pathogens detection method that sensitive, specificity is high.
In order to solve the problems of the technologies described above, the present invention proposes following technical scheme: the aptamer of fluorescence probe mark can detect stronger fluorescence signal under free state, when aptamer is adsorbed on metal-organic framework materials, fluorescence is quenched, object bacteria is added in system, object bacteria can be combined with aptamer specifically, and aptamer is separated from metal-organic framework materials, and then causes fluorescence signal to strengthen.In theory, the concentration of object bacteria is higher, and the aptamer separated is more, and the amount of corresponding fluorescence probe is more, and fluorescence signal is stronger, and can carry out quantitative test accordingly, Cleaning Principle as depicted in figs. 1 and 2.
In the above-mentioned method based on metal-organic framework materials-aptamer fluorescent optical sensor detection food-borne pathogens, described aptamer base sequence is obtained by document or patent.The aptamer sequence wherein with salmonella with high-affinity and high specific is 5 '-TTTG GTCC TTGT CTTA TGTC CAGA ATGC TATG GCGG CGTC ACCC GACG GGGA CTTG ACAT TA-3 ' (Mol Cell Probe, 2009,23:20), at the upper fluorescence probe Fluoresceincarboxylic acid (FAM) of 3 ' end mark, completed by Sangon Biotech (Shanghai) Co., Ltd..
In the above-mentioned method based on metal-organic framework materials-aptamer fluorescent optical sensor detection food-borne pathogens, described metal-organic framework materials has fluorescent quenching characteristic and the adsorbability to aptamer, prepares by methods such as microwave method, ultrasonic method, synthesis in solid state and electrochemistry formated.Preparation flow wherein containing Cd organic framework material is as follows:
Step 1: by moisture caddy (CdCl
22.5H
2o, 0.023g ~ 0.115g, 0.1mmol ~ 0.5mmol) and trimesic acid (H
2bTC, 0.042g ~ 0.126g, 0.2mmol ~ 0.6mmol) be dissolved in the N of 5.0mL ~ 15.0mL, in the cyclohexanol of N-dimethyl formamide (DMF), 1.0mL ~ 5.0mL and 1.0mL ~ 5.0mL water, after mixing, at room temperature stir 3 ~ 6 hours, for subsequent use.
Step 2: step 1 gained solution is transferred in beaker, stir while slowly drip the triethylamine of 0.1mL ~ 0.6mL, within 10 minutes ~ 20 minutes, dropwise, seal large beaker with plastic sheeting, beaker is put into ultrasonic reactor, at 40 DEG C ~ 70 DEG C ultrasonic 10 minutes ~ 30 minutes.Product is taken out centrifugal filtration, and by 50mL ~ 100mL DMF solvent repeatedly washed product, at being placed in 50 DEG C ~ 70 DEG C, vacuum drying 24 hours ~ 36 hours, namely obtains [Cd
4(BTC)
3(DMF)
2(H2O)
2] porous is containing Cd organic framework material.
Compared with existing pathogenic microbes detect technology, remarkable advantage of the present invention is:
(1) the fluorescent quenching characteristic of metal-organic framework materials and the adsorbability to aptamer is utilized, metal-organic framework materials is applied to Food Safety Analysis field, the high-affinity that bind nucleic acid is fit and high specific recognition capability, for the quick Detection and Identification of food-borne pathogens provides effective means, be expected to form new Food Safety Analysis method.
(2) the inventive method is a kind of detection method of general character, not only can be widely used in the detection of pathogenic bacteria, also can be applicable to the detection of biomacromolecule (nucleic acid, protein) and Small molecular (in food and environment chemical pollutant etc.).
Accompanying drawing explanation
Fig. 1 detects the schematic diagram of pathogenic bacteria based on metal-organic framework materials-aptamer fluorescent optical sensor.
Fig. 2 corresponds to the fluorescence spectrum figure in Fig. 1 under a, b, c tri-kinds of states.
Fig. 3 contains fluorescence spectrum figure and the working curve (illustration) of Cd organic framework material-FAM labeling nucleic acid fit detection variable concentrations salmonella.
Fig. 4 is containing the fit signal contrast figure detecting salmonella and other pathogenic bacteria of Cd organic framework material-FAM labeling nucleic acid, and wherein the concentration of salmonella is 5.5 × 10
3cfu/mL, the concentration of other bacterium is 5.5 × 10
4cfu/mL.
Embodiment
Example below will specifically introduce method of operating of the present invention, but can not as limitation of the invention.
Embodiment 1: the inventive method is detecting the application in salmonella
First synthesis is containing Cd organic framework material, and flow process is as follows:
Step 1: by CdCl
22.5H
2o(0.046g, 0.2mmol) and H
2bTC(0.105g, 0.5mmo) be dissolved in DMF, 3.0mL of 8.0mL cyclohexanol and 2.0mL water in, at room temperature stir 4 hours after mixing, for subsequent use.
Step 2: be transferred in beaker by step 1 gained solution, stirs the triethylamine slowly dripping 0.2mL, within 10 minutes, dropwises, seal large beaker with plastic sheeting, beaker is put into ultrasonic reactor, at 60 DEG C ultrasonic 15 minutes.Product is taken out centrifugal filtration, and by 60mL DMF washed product, at being placed in 70 DEG C, vacuum drying 24 hours, namely obtains [Cd
4(BTC)
3(DMF)
2(H2O)
2] porous is containing Cd organic framework material.
By above-mentioned obtained evenly grinding containing Cd organic framework material, take the powder 1.0mg sonic oscillation after grinding and be dispersed in 1.0mL distilled water, obtain 1.0mg/mL containing Cd material solution, for subsequent use at being stored in 4 DEG C.
The detection of Salmonella aptamer 1.0nmol getting FAM mark is dissolved in 1.0mL distilled water, and obtained concentration is 1.0nmol/mL aptamer solution, and stored protected from light is for subsequent use at 4 DEG C.
Get 20 μ L(1.0mg/mL respectively) containing Cd organic framework material solution, 10 μ L(1.0nmol/mL) aptamer solution, two solution are joined 180 μ L Tris-HCl(pH 7.4) in buffer solution, keep 30 minutes at 37 DEG C, detect fluorescence signal.
In above-mentioned mixed solution, add the salmonella of 10 μ L variable concentrations, at 37 DEG C, hatch 1 hour, detect the fluorescence signal under the existence of variable concentrations detection of Salmonella.
Result shows, when salmonella concentration is 18 ~ 3.2 × 10
4during cfu/mL concentration range, fluorescence signal and log concentration be relevant (y=46.15x+14.27, R=0.9963) linearly, and detection limit reaches 5 cfu/mL(S/N=3), as shown in Figure 3.
Embodiment 2: the inventive method is detecting the application in salmonella and other pathogenic bacteria
For evaluating the specificity of the inventive method, choose food-borne pathogens common in food as salmonella, staphylococcus aureus, Escherichia coli, Shigella and listeria spp, according to the detecting step described in embodiment 1, these pathogenic bacteria are measured, record respective fluorescence signal and contrasted, result as shown in Figure 4.Object bacteria due to aptamer is salmonella, therefore salmonella (5.5 × 10
3cfu/mL) fluorescence signal is the strongest, and other pathogenic bacteria when concentration 10 doubly to salmonella, fluorescence intensity is the 1/10th even lower of salmonella signal.This result shows that the method detecting pathogenic bacteria based on metal-organic framework materials-aptamer fluorescent optical sensor has high degree of specificity and selectivity.
Embodiment 3: the inventive method is applied to the detection of salmonella in beef sample
Fresh beef freezing for 10g is rubbed, with 100mL Tris-HCl(pH 7.4) buffer solution homogeneous 15 minutes, cross and filter bulky grain and suspension, get supernatant for subsequent use, measure the salmonella in supernatant according to the detecting step described in embodiment 1.Get 5 parts of beef sample Parallel testings.
The salmonella (1.0 × 10 of variable concentrations is injected respectively in the supernatant of 5 parts of beef samples
3~ 5.0 × 10
3cfu/mL), each concentration all confirms through colony counting method, and then detect the salmonella in sample according to the detecting step described in embodiment 1, testing result is as shown in table 1 again.All do not detect salmonella in 5 parts of beef samples, the relative standard deviation (RSD) of mark-on experiment is between 3.6% ~ 7.5%, and the recovery is in 90.0% ~ 106.0% scope.As can be seen here, the inventive method has higher accuracy and precision, is applicable to actual sample analysis.
Table 1 is containing salmonella in the fit detection beef sample of Cd organic framework material-FAM labeling nucleic acid
A. ND: do not detect;
B. mean value ± standard deviation,
n=5.
Claims (2)
1. one kind is detected the method for food-borne pathogens based on metal-organic framework materials-aptamer fluorescent optical sensor, it is characterized in that: the detecting pattern strengthened after devising the first cancellation of a kind of fluorescence, first the aptamer of fluorescence probe mark is adsorbed on metal-organic framework materials, fluorescence probe is quenched, object bacteria is added in system, the aptamer of fluorescence probe mark leaves metal-organic framework materials and is combined with object bacteria, thus fluorescence probe signal is strengthened, realize the detection to salmonella in sample according to the detection of fluorescence signal.
2. a kind of method detecting food-borne pathogens based on metal-organic framework materials-aptamer fluorescent optical sensor according to claim 1, it is characterized in that: described metal-organic framework materials not only has the function of efficient adsorption aptamer, and there is good fluorescent quenching characteristic.
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| CN106018360A (en) * | 2016-05-13 | 2016-10-12 | 深圳职业技术学院 | Method for detecting urea based on metal organic framework material fluorescent sensor |
| CN110006971A (en) * | 2019-03-12 | 2019-07-12 | 宁波大学 | A kind of preparation method and applications of the aptamer sensor of binary channels output detection food-borne pathogens |
| CN110384685A (en) * | 2019-09-04 | 2019-10-29 | 临沂大学 | A kind of metal organic frame pharmaceutical carrier and preparation method thereof of nucleic acid modification |
| WO2022179183A1 (en) * | 2021-02-25 | 2022-09-01 | 东南大学 | Nucleic acid functionalized metal nanoprobe and preparation method therefor |
| CN115057472A (en) * | 2022-06-21 | 2022-09-16 | 中国医学科学院基础医学研究所 | Novel fluorescence sensing system and application thereof in PTP-1B detection |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106018360A (en) * | 2016-05-13 | 2016-10-12 | 深圳职业技术学院 | Method for detecting urea based on metal organic framework material fluorescent sensor |
| CN106018360B (en) * | 2016-05-13 | 2018-07-17 | 深圳职业技术学院 | A method of urea is detected based on metal-organic framework materials fluorescent optical sensor |
| CN110006971A (en) * | 2019-03-12 | 2019-07-12 | 宁波大学 | A kind of preparation method and applications of the aptamer sensor of binary channels output detection food-borne pathogens |
| CN110006971B (en) * | 2019-03-12 | 2021-03-09 | 宁波大学 | Preparation method and application of aptamer sensor for detecting food-borne pathogenic bacteria through dual-channel output |
| CN110384685A (en) * | 2019-09-04 | 2019-10-29 | 临沂大学 | A kind of metal organic frame pharmaceutical carrier and preparation method thereof of nucleic acid modification |
| WO2022179183A1 (en) * | 2021-02-25 | 2022-09-01 | 东南大学 | Nucleic acid functionalized metal nanoprobe and preparation method therefor |
| CN115057472A (en) * | 2022-06-21 | 2022-09-16 | 中国医学科学院基础医学研究所 | Novel fluorescence sensing system and application thereof in PTP-1B detection |
| CN115057472B (en) * | 2022-06-21 | 2023-10-27 | 中国医学科学院基础医学研究所 | Novel fluorescence sensing system and application thereof in PTP-1B detection |
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Application publication date: 20151104 |