CN116773442A - Rapid photochromic multi-signal detection kit and method for food-borne pathogenic bacteria - Google Patents
Rapid photochromic multi-signal detection kit and method for food-borne pathogenic bacteria Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/3103—Atomic absorption analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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Abstract
The invention discloses a rapid colorimetric/fluorescent detection kit and a rapid colorimetric/fluorescent detection method for food-borne pathogenic bacteria, wherein the rapid colorimetric/fluorescent detection kit comprises the following components: an aptamer solution of food-borne pathogenic bacteria, a colloidal gold solution, a fluorescein isothiocyanate solution, a negative quality control product and a positive quality control product. The method realizes the specific identification of food-borne pathogenic bacteria through simple centrifugation, utilizes the food-borne pathogenic bacteria aptamer to induce colloidal gold to undergo aggregation change after freeze thawing, quantitatively detects staphylococcus aureus according to the aggregation color change of colloid and the influence of fluorescein isothiocyanate, has small variation coefficient during quantitative detection, and has the advantages of high sensitivity, wide linear range, low cost, simple operation, easy implementation, short detection time, 5 CFU/mL colorimetric signal detection limit, 2 CFU/mL fluorescent quantitative detection limit, and double-signal on-site visualization and detection accuracy improvement.
Description
Technical Field
The invention belongs to the technical field of biosensing, and particularly relates to a rapid photochromic multi-signal detection kit and method for food-borne pathogenic bacteria.
Background
Bacterial contamination seriously affects human health, which is an important issue of concern in the public health field. Bacteria are widely existing in nature, such as water, air, dust, human and animal excreta, and can enter human bodies through the ways of spray, contact, drinking water, food and the like, so that the incidence rate of infection is high, and the human health is threatened greatly. Among them, food-borne pathogenic bacteria pose a serious threat to human health as biological contaminants in foods. According to the world health organization, about 6 million people worldwide are poisoned or diseased by food pollution each year, and 42 ten thousand people die from food poisoning, one third of which is children under five years old. Food-borne pathogenic bacteria which are specified in national standard "limit of pathogenic bacteria in food" and which are necessary to be detected by food include staphylococcus aureus, salmonella, vibrio parahaemolyticus, listeria monocytogenes and escherichia coli O157: H7. These standards and specifications provide a guarantee for food safety and crowd health, but preventing food contamination is a persistent challenge for the global health care system and the food industry, and it is significant to be able to establish a rapid, sensitive and accurate detection method for detecting pathogenic bacteria and to make countermeasures in time on the basis of this.
At present, detection methods for pathogenic bacteria mainly comprise a separation culture method, a molecular biological detection method, an immunological method and the like, and although the methods can realize the detection of food-borne pathogenic bacteria, the respective defects still exist, for example, the separation culture method is a gold standard for food-borne detection and is also a method recommended in national standards of China, but the experiment period is long, the operation is complex, and the requirement for rapid detection of samples cannot be met; other detection methods, such as molecular biology detection methods, are complex in technical operation, high in cost and require specialized staff; the enzyme-linked immunosorbent assay requires the use of biological enzymes, which are expensive and whose activity is susceptible to environmental factors such as temperature, pH and the like. Therefore, there is an urgent need to develop a field detection method that is simple to operate, easy to implement, fast and highly sensitive.
One of the key problems in detecting food-borne pathogens is to realize on-site rapid screening of a large number of samples. Visual colorimetric analysis is the best on-site rapid signal screening method. The colloidal gold material has good biocompatibility, large specific surface area, photoelectric property and surface plasmon resonance effect. The molar absorptivity of the ultraviolet-visible light-absorbing material gradually decreases along with the increase of the particle size, and the ultraviolet-visible absorption peak shifts blue and shows macroscopic color change. Therefore, colloidal gold is widely used in on-site visual colorimetric sensing detection. However, the visual colorimetry has a single signal and low sensitivity, and thus cannot realize quantitative detection. The multiple signal analysis method is a novel detection method with self-correction and self-verification (Fu, x., sun, j., ye, y., zhang, y., sun, x. (2022). A rapid and ultrasensitive dual detection platform based on Cas 12-a for simultaneous detection of virulence and resistance genes of drug-resistive Salmonella, biosensors and Bioelectronics, 195, 113682.). The detection system can generate two or more optical, electric and color signals through one-time reaction, the signals can be corrected and mutually referred, and the defects of a visual colorimetric analysis method can be overcome and the detection system is used for field practice. Fluorescein isothiocyanate is an important fluorescent dye molecule, is low in cost, high in extinction coefficient of light and good in fluorescence quantum yield, and is widely used for protein fluorescent labeling. The nucleic acid aptamer is an oligonucleotide fragment screened by using an in vitro index enrichment ligand system evolution technology, can be combined with a plurality of target substances in a high specificity and high selectivity way, and has a great application prospect in the detection and analysis fields of medicine, biology, environment, agriculture and forestry and the like.
In summary, in order to realize on-site screening of food-borne pathogenic bacteria, a detection method and a kit which are rapid, simple, high in accuracy and strong in specificity are developed, and have important significance in the fields of food microorganism detection and the like.
Disclosure of Invention
The invention aims to provide a method and a kit for detecting multiple light color signals of food-borne pathogenic bacteria.
A rapid photochromic multi-signal detection kit for food-borne pathogenic bacteria comprises: a pathogenic bacteria aptamer solution, a colloidal gold solution and a fluorescein isothiocyanate solution;
the concentration of the food-borne pathogenic bacteria aptamer solution is 10 mu M-30 mu M; the colloid Jin Lijing is 13-40 nm, and the surface functional group is carboxyl; the concentration of the fluorescein isothiocyanate solution is 2.5-20 mu M;
the food-borne pathogenic bacteria comprise: staphylococcus aureus, listeria coli;
the staphylococcus aureus aptamer gene sequence is 5'SH-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTG CTA A-3'.
The colloidal gold solution is prepared by reacting chloroauric acid and trisodium citrate.
The reaction was carried out by heating chloroauric acid of 1 mM to boiling, rapidly adding trisodium citrate of 4 mmol/L with vigorous stirring, and reacting for 20 min.
The concentration of the aptamer solution is 20 mu M; the preferred concentration of fluorescein isothiocyanate FITC is 15. Mu.M; the particle size of the colloidal gold is 13 nm.
The kit also comprises negative quality control phosphate buffer solution and positive quality control inactivated staphylococcus aureus suspension.
A rapid colorimetric/fluorescent detection method for food-borne pathogenic bacteria, comprising: a rapid colorimetric/fluorescent detection kit for food-borne pathogenic bacteria according to claim 1;
1) Adding a sample to be detected into a buffer solution, fully and uniformly mixing, and transferring the leaching solution into a centrifuge tube;
the buffer solution has a volume of 5-10 mL, preferably a volume of 5 g sample plus buffer 10 mL;
2) Adding food-borne pathogenic bacteria aptamer solution, mixing with vortex, incubating at 37deg.C for 15-90 min, centrifuging, and sucking supernatant with a pipette;
3) Adding the supernatant solution into colloidal gold solution at a temperature of-20 to-80 DEG C o Freezing for 10-60 min at 25-70 o After melting under C, visually observing or measuring the scanning spectrum of the colloidal gold solution at 400-800 nm by an ultraviolet-visible spectrometer;
4) Centrifuging the dissolved colloidal gold solution to remove aggregated nano particles, adding a fluorescein solution for incubation, measuring fluorescence by using a fluorescence photometer, and determining the content of food-borne pathogenic bacteria.
The freezing temperature in the step 3) is-80 o C, the time is 10min, and the melting temperature is 70 o C。
The incubation time in step 2) was 60 min.
The invention provides a rapid colorimetric/fluorescent detection kit and a rapid colorimetric/fluorescent detection method for food-borne pathogenic bacteria, wherein the rapid colorimetric/fluorescent detection kit comprises the following components: an aptamer solution of food-borne pathogenic bacteria, a colloidal gold solution, a fluorescein isothiocyanate solution, a negative quality control product and a positive quality control product. The method realizes the specific identification of food-borne pathogenic bacteria through simple centrifugation, utilizes the food-borne pathogenic bacteria aptamer to induce colloidal gold to undergo aggregation change after freeze thawing, quantitatively detects staphylococcus aureus according to the aggregation color change of colloid and the influence of fluorescein isothiocyanate, has small variation coefficient during quantitative detection, and has the advantages of high sensitivity, wide linear range, low cost, simple operation, easy implementation, short detection time, 5 CFU/mL colorimetric signal detection limit, 2 CFU/mL fluorescent quantitative detection limit, and double-signal on-site visualization and detection accuracy improvement.
Drawings
FIG. 1 is a transmission electron microscope image of gold nanoparticles;
FIG. 2 (a) ultraviolet spectrum of colloidal gold; (b) Fluorescence emission spectra of Fluorescein Isothiocyanate (FITC);
FIG. 3 (a) is an absorption spectrum of colloidal gold of different particle sizes; (b) The influence of freeze thawing on the spectrum of the mixed solution of colloidal gold and the aptamer with different particle diameters; (c) absorbance normalization after colloid Jin Dongrong;
fig. 4 (a, b) colloid Jin Mogu mycin mix indicates optimization of liquid cooling time;
FIG. 5 (a-c) optimization of dissolution temperature of colloidal Jin Mogu mycin mixing indicator;
FIG. 6 (a) Staphylococcus aureus aptamer with Staphylococcus aureus incubation time optimization; (b) optimizing the concentration of staphylococcus aureus aptamer; (c) optimization of fluorescein concentration;
FIG. 7 standard curve, linear regression equation and correlation coefficients for Staphylococcus aureus detection;
FIG. 8 selectivity of Staphylococcus aureus detection;
Detailed Description
EXAMPLE 1 preparation of gold nanoparticles (colloidal gold)
Firstly, soaking glassware required by synthesis in aqua regia (nitric acid: hydrochloric acid=1:3) for 30min, flushing with ultrapure water for three times, drying with nitrogen, heating chloroauric acid with the concentration of 1 mM of 100 mL to boiling, rapidly adding trisodium citrate of 4 mM in a stirring state, changing the solution from light yellow to wine red after 20min, obtaining gold nanoparticles, cooling to room temperature, and preserving in dark condition at 4 ℃. The uv spectrum of the gold nanoparticles is shown in fig. 2 (a).
Example 2 preparation of phosphate buffer (dilution for bacteria and negative control)
Buffer solution: taking NaCl 8.0 g, KCl 0.2 g and Na 2 HPO 4 1.44 g and KH 2 PO 4 0.24 g is dissolved in 800 mL distilled water, pH 7.4 is adjusted by NaOH, and the volume is fixed to 1000 mL.
EXAMPLE 3 preparation of Fluorescein Isothiocyanate (FITC) solution
Accurately weighing 0.0019 g of FITC, dissolving in deionized water at room temperature, and diluting to form 2.5 mu M-20 mu M of FITC solution. FITC fluorescence emission spectra are shown in FIG. 2 (b).
EXAMPLE 4 preparation of Staphylococcus aureus aptamer solution
Dissolving the aptamer (5 'SH-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTG CTA A-3') dry powder in enzyme-free water (water without DNA or RNase) to form an aptamer solution of 10 mu M-30 mu M;
example 5 gold nanoparticle particle size optimization experiments
Gold nanoparticles with the particle diameters of 13nm, 20nm and 40nm are prepared by adopting a sodium citrate reduction method, and ultraviolet absorption spectra are respectively measured by an ultraviolet-visible spectrophotometer; then 100 mu L of gold nanoparticle solution with each particle size is accurately removed, 25 mu L of supernatant of negative control group is added into the solution, the solution is frozen at-80 ℃ for 10min, thawed at 70 ℃, and ultraviolet absorption spectrum is scanned.
As shown in FIG. 3, it can be seen from FIG. 3 (a) and FIG. 3 (b) that the absorption peaks of gold nanoparticles with different particle diameters are changed between 520nm and 531 nm; from the graph (c), it can be seen that the 20nm and 40nm gold nanoparticles exhibited an aggregated state after freeze thawing, while the 13nm gold nanoparticles were hardly changed. Therefore, 13nm gold nanoparticles are preferred for this experiment.
Example 6 gold nanoparticle and aptamer freezing time optimization experiment
Taking 100 mu L of gold nanoparticle solution, adding 25 mu L of negative control group supernatant, uniformly mixing the solution, standing at-80 ℃ for 5min, 10min, 20min, 30min and 60min, and thawing at 70 ℃. And measuring the ultraviolet absorption spectrum of the mixed solution by an ultraviolet-visible spectrophotometer.
As shown in FIG. 4 (a) and (b), it can be seen from FIG. 4 (a) and (b) that gold nanoparticles slightly aggregated when frozen at-80℃for 5min, and that gold nanoparticles were similar in state when frozen for more than 10min. The invention preferably freezes at-80 ℃ for 10min.
Example 7 gold nanoparticle and aptamer melting temperature optimization experiment
Taking 100 mu L of gold nanoparticle solution, adding 25 mu L of negative control group supernatant, uniformly mixing the solution, freezing at-80 ℃ for 10min, thawing at 20 ℃, 37 ℃,50 ℃, 60 ℃ and 70 ℃, and measuring the ultraviolet absorption spectrum of the mixed solution by an ultraviolet-visible spectrophotometer.
As shown in fig. 5 (a) (b) (c), it can be seen from fig. 5 (a) (b) (c) that the melting temperature does not affect the aggregation of gold nanoparticles, and the absorption peak hardly changes, but the time required for thawing gradually decreases with increasing melting temperature. The invention preferably thaws rapidly at 70 ℃.
Example 8 Staphylococcus aureus aptamer and time of incubation optimization experiment
mu.L of 20. Mu.M Staphylococcus aureus aptamer was combined with 100. Mu.L of 10 5 After evenly mixing CFU/mL staphylococcus aureus, placing in 37 ℃ for incubation for 15 min, 30min, 45 min, 60min and 90 min, centrifuging at 4000 rpm for 10min, taking 25 mu L of supernatant and 100 mu L of gold nanoparticle solution, evenly mixing, freezing at-80 ℃ for 10min, and measuring the ultraviolet absorption spectrum of the mixed solution by an ultraviolet-visible spectrophotometer after melting.
As shown in FIG. 6 (a), the test results show that A shows that the incubation time increases 650 /A 520 Gradually increasing; when the incubation time is 60min, A 650 /A 520 The increase was smoothed, so the incubation time for this experiment was preferably 60 min.
Example 9 Staphylococcus aureus aptamer concentration optimization experiment
Staphylococcus aureus aptamers at concentrations of 10. Mu.L at 0. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 25. Mu.M and 30. Mu.M, respectively, were combined with 100. Mu.L of 10 5 CFU/mL staphylococcus aureus was mixed as a positive control group. Mixing 10 mu L of staphylococcus aureus aptamer (0 mu M, 10 mu M, 15 mu M, 20 mu M, 25 mu M and 30 mu M) with 100 mu L of phosphoric acid buffer solution to serve as a negative control group, uniformly mixing the solution, incubating at 37 ℃ for 60min, centrifuging at 4000 rpm for 10min, uniformly mixing 25 mu L of supernatant with 100 mu L of gold nanoparticle solution, freezing at-80 ℃ for 10min, and measuring the ultraviolet absorption spectrum of the mixed solution by an ultraviolet-visible spectrophotometer after melting.
As shown in FIG. 6 (b), when the concentration of the Staphylococcus aureus aptamer added was 20. Mu.M, A was found to be present or absent 650 /A 520 The difference is the largest, a more pronounced colorimetric signal can be obtained, and therefore the preferred concentration of aptamer is 20 μm.
EXAMPLE 10 Fluorescein Isothiocyanate (FITC) concentration optimization experiments
mu.L of Staphylococcus aureus aptamer at a concentration of 20. Mu.M was mixed with 100. Mu.L of a solution containing 10 5 CFU/mL staphylococcus aureus was incubated at 37℃for 1 h. After the mixed solution is centrifuged at 4000 rpm for 10min, 25. Mu.L of supernatant is added to gold nanoparticles with a volume of 100. Mu.L, and the mixture is put into a refrigerator for freeze thawing. Centrifuging the frozen and thawed gold nanoparticles at 4000 rpm for 10min, and incubating 90 mu L of the centrifuged AuNPs solution with 10 mu L of FITC with concentrations of 2.5,5, 10, 15 and 20 mu M at room temperature for 10 min; the fluorescence spectra of the above mixed solutions were measured separately in the negative control group using a phosphate buffer instead of staphylococcus aureus using a fluorescence photometer, and a fluorescence difference Δf was calculated.
As shown in FIG. 6 (c), the difference in fluorescence was maximum when the concentration of FITC added was 15. Mu.M. Thus, the preferred concentration of fluorescein isothiocyanate FITC is 15. Mu.M.
EXAMPLE 11 staphylococcus aureus assay
Under the optimal condition, the established colorimetric/fluorescent dual-mode detection method for staphylococcus aureus comprises the following steps of:
(1) Dilution of staphylococcus aureus solution
Taking 6 microcentrifuge tubes of 1.5 mL, respectively numbering 1,2,3,4,5 and 6, adding 1000 mu L of standard staphylococcus aureus solution into a tube 1, adding 900 mu L of standard diluent (phosphate buffer solution) into a tube 2-6, repeatedly blowing with a gun head for about 5-10 times (taking the attention that bubbles are easy to generate if the control amplitude is not too large), and if a vortex instrument is arranged, vortex for about 1-10 seconds on the vortex instrument, and then replacing the gun head. 100 μl was added to tube No. 2 in tube No. 1, and the procedure was followed by analogy. After final dilution, the liquid in the 1 st to 5 th tubes was 900. Mu.L and the liquid in the 6 th tube was 1000. Mu.L. The concentration of staphylococcus aureus is from big to small, and sequentially 1×10 6 -10 CFU/mL。
(2) Staphylococcus aureus aptamer binding to staphylococcus aureus
Mixing 10 μL of Staphylococcus aureus aptamer with concentration of 20 μM with 10-10 6 Mixing staphylococcus aureus with CFU/mL concentration, incubating at 37 ℃ for 60min, centrifuging at 4000 rpm for 10 min;
(3) Addition of colloidal gold solution
Adding 25 mu L of the supernatant obtained in the step (2) into 100 mu L of gold nanoparticle solution, freezing at-80 ℃ for 10min, and thawing;
(4) Color development
And indicating the content of staphylococcus aureus according to the color and aggregation state of the unfrozen colloidal gold solution.
(5) Addition of fluorescein isothiocyanate FITC solution
Centrifuging the unfrozen colloidal gold solution at 4000 rpm for 10min, and incubating 90 mu L of colloidal gold solution with 10 mu L of 15 mu M fluorescein isothiocyanate FITC solution;
(6) Reading the number
The ultraviolet absorbance spectra were scanned with an ultraviolet-visible spectrophotometer at 400-800 nm, recording the absorbance intensities at 520nm and 650 nm; recording the emission spectrum of 500-600 nm with a fluorescence photometer under 490 nm excitation light; the concentration of staphylococcus aureus was determined based on standard curve, negative sample correction.
The detection results of staphylococcus aureus using the colorimetric/fluorescent method (absorbance ratio and fluorescent signal) of the present invention are shown in fig. 7, wherein fig. 7 (a) is a scanning spectrum when staphylococcus aureus of different concentrations is detected by freeze thawing of gold nanoparticles and an aptamer, and a linear calibration curve is shown in fig. 7 (b). The colorimetric method has a linear detection range of 10 for staphylococcus aureus 2 -10 6 CFU/mL, the linear relation is good, the correlation coefficient is 0.994, and the detection lower limit is 5 CFU/mL.
FIG. 7 (c) is a fluorescence curve of detection of Staphylococcus aureus at different concentrations based on the action of gold nanoparticles and fluorescein isothiocyanate FITC, and the linear calibration curve is shown in FIG. 7 (d). The fluorescence method has a linear detection range of 10 for staphylococcus aureus 1 -10 6 CFU/mL, the linear relation is good, the correlation coefficient is 0.991, and the detection lower limit is 2 CFU/mL.
EXAMPLE 12 specificity experiments
Accurately remove 100 mu L of concentration 10 respectively 5 CFU/mL E.coli O157H 7 at a concentration of 10 5 CFU/mL Pseudomonas, concentration 10 5 CFU/mL Listeria monocytogenes at a concentration of 10 5 Adding mixed bacteria of CFU/mL staphylococcus aureus and the bacteria into each tube sequentially, adding 10 mu L of 20 mu M staphylococcus aureus aptamer into each tube, uniformly mixing the solutions, incubating at 37 ℃ for 60min, centrifuging at 4000 rpm for 10min, adding 25 mu L of supernatant into 100 uL gold nanoparticle solution, freezing at-80 ℃ for 10min, thawing at room temperature, and measuring the absorption value; the thawed colloidal gold solution was centrifuged at 4000 rpm for 10min, and 90. Mu.L of the colloidal gold solution was incubated with 10. Mu.L of 15. Mu.M fluorescein isothiocyanate solution to measure fluorescence spectrum thereof.
The experimental results are shown in FIG. 8, and it can be seen from FIG. 8 that the method can specifically recognize Staphylococcus aureus. Therefore, the colorimetric/fluorescent dual-mode detection method for staphylococcus aureus based on aptamer-induced colloidal gold aggregation and the establishment of the effect of gold nanoparticles on fluorescein isothiocyanate has the advantages of high detection selectivity and good specificity.
EXAMPLE 13 preparation and use of Staphylococcus aureus kit
A rapid detection kit for staphylococcus aureus, comprising: from the colloidal gold described in example 1, the phosphate buffer described in example 2, the fluorescein isothiocyanate solution described in example 3, 10 inactivated with 1% formaldehyde 9 Bacterial liquid positive standard and negative quality control phosphate buffer.
The rapid detection kit for staphylococcus aureus comprises the following steps:
grinding a food sample to be detected, taking 5 g homogenate, adding a phosphate buffer solution 10 mL in the kit, fully mixing, transferring 100 mu L of leaching solution into a centrifuge tube, adding 10 mu L of aptamer in the kit into the centrifuge tube, incubating for 60min at 37 ℃ after vortex mixing, centrifuging at 4000 rpm for 10min, and sucking out the supernatant by using a pipette. Taking supernatant 25. mu.L was added to 100. Mu.L of the gold nanoparticle solution, which was frozen at-80℃for 10 minutes, thawed, and the absorption value was measured. The absorbance was measured by a visible spectrophotometer and also visually observed. The thawed colloidal gold solution was centrifuged at 4000 rpm for 10min, and 90. Mu.L of the colloidal gold solution was incubated with 10. Mu.L of 15. Mu.M fluorescein isothiocyanate FITC solution to measure fluorescence spectrum thereof. Detecting negative quality control sample and positive quality control sample provided in the kit by the same method, and preparing standard curve by using standard bacterial liquid, wherein A is positive quality control sample 650 /A 520 And if the fluorescence value of the sample smaller than the negative quality control product or the positive quality control product is smaller than the negative quality control product, the kit is invalid.
Claims (10)
1. A rapid colorimetric/fluorescent detection kit for food-borne pathogenic bacteria, comprising: an aqueous solution of food-borne pathogenic bacteria aptamer, a colloidal gold solution, and a fluorescein isothiocyanate solution;
the concentration of the staphylococcus aureus aptamer solution is 10 mu M-30 mu M; the colloid Jin Lijing is 13-40 nm, and the surface functional group is carboxyl; the concentration of the fluorescein isothiocyanate solution is 2.5-20 mu M.
2. The rapid colorimetric/fluorescent detection kit for food-borne pathogenic bacteria of claim 1, wherein: the food-borne pathogenic bacteria is staphylococcus aureus, and the aptamer gene sequence is 5'SH-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTG CTA A-3'.
3. A food borne pathogenic bacteria rapid colorimetric/fluorescent detection kit according to claims 1 and 2, characterized in that: the colloidal gold solution is prepared by reacting chloroauric acid and trisodium citrate.
4. A food borne pathogenic bacteria rapid colorimetric/fluorescent detection kit according to claim 3, wherein: the reaction was carried out by heating chloroauric acid of 1 mM to boiling, rapidly adding trisodium citrate of 4 mmol/L with vigorous stirring, and reacting for 20 min.
5. A food borne pathogenic bacteria rapid colorimetric/fluorescent detection kit according to claim 3, wherein: the concentration of the aptamer solution is 20 mu M; the concentration of fluorescein isothiocyanate FITC is 15 mu M; the particle size of the colloidal gold is 13 nm.
6. The rapid colorimetric/fluorescent detection kit for food-borne pathogenic bacteria of claim 5, wherein: the kit also comprises a negative quality control product: phosphate buffer, positive quality control: inactivating the staphylococcus aureus suspension.
7. A rapid colorimetric/fluorescent detection method for food-borne pathogenic bacteria, comprising: a rapid colorimetric/fluorescent detection kit for food-borne pathogenic bacteria according to claim 1;
1) Adding a sample to be detected into a buffer solution, fully and uniformly mixing, and transferring the leaching solution into a centrifuge tube;
the buffer solution has a volume of 5-10 mL, preferably a volume of 5 g sample plus buffer 10 mL;
2) Adding staphylococcus aureus aptamer solution, uniformly mixing by vortex, incubating for 15-90 min at 37 ℃, centrifuging, and sucking out supernatant;
3) Adding the supernatant solution into colloidal gold solution at a temperature of-20 to-80 DEG C o Freezing for 10-60 min at 25-70 o After melting under C, visually observing or measuring the scanning spectrum of the colloidal gold solution at 400-800 nm by an ultraviolet-visible spectrometer;
4) Centrifuging the dissolved colloidal gold solution, removing aggregated nano particles, adding a fluorescein solution for incubation, measuring fluorescence by using a fluorescence photometer, and determining the content of staphylococcus aureus.
8. A rapid colorimetric/fluorescent detection method for food-borne pathogenic bacteria is characterized in that: the freezing temperature in the step 3) is-80 o C, the time is 10min, and the melting temperature is 70 o C。
9. A rapid colorimetric/fluorescent detection method for food-borne pathogenic bacteria is characterized in that: the incubation time in step 2) was 60 min.
10. The rapid colorimetric/fluorescent detection kit for food-borne pathogenic bacteria of claim 6, wherein: the buffer solution is prepared from NaCl 8.0 g, KCl 0.2 g and Na 2 HPO 4 1.44 g and KH 2 PO 4 0.24 g is dissolved in 800 mL distilled water, pH 7.4 is adjusted by NaOH, and the volume is fixed to 1000 mL.
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