A bacteriophage bioluminescent array bacterial detection chip and its preparation method and application

Abstract

The present invention provides a bacteriophage bioluminescent array bacteria detection chip and a preparation method and application thereof, belonging to the technical field of microbial content detection in food. The present invention uses polydiallyldimethylammonium chloride, polylactic acid and LB culture medium to prepare a bacteriophage bioluminescent array bacteria detection chip substrate, and sequentially performs bacteria modification, bacteriophage modification and luminescence modification to obtain a bacteriophage bioluminescent array bacteria detection chip. The LB culture medium attracts bacteria to grow on the surface of a reaction tank; the polymer substance polydiallyldimethylammonium chloride with positively charged groups on the surface firmly adsorbs the bacteriophage with negatively charged heads on the surface of the reaction tank. The chip prepared by the present invention has high sensitivity, high accuracy and strong specificity, and the drug sensitivity test time for toxic strains is shortened to within 2 hours, and the preparation process is simple, no large-scale equipment is required, the reaction conditions are mild, the cost is low and the safety factor is high.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial content detection in food, and in particular to a bacteriophage bioluminescent array bacteria detection chip and a preparation method and application thereof. Background Art

[0002] Outbreaks of foodborne diseases caused by microbial pathogens are very common around the world. Moreover, there is obviously more than one type of bacteria on the surface of food, and the bacterial concentration can be extremely complex and variable due to environmental reasons. Among the existing microbial content detection technologies such as direct counting method, turbidimetry, live bacteria counting method, gravimetric method, total cell nitrogen or total carbon method, and color change unit method, direct counting method, live bacteria counting method, and gravimetric method have obvious, huge and difficult to eliminate systematic errors in the determination of bacterial numbers; the turbidimetric method has a high detection limit and is difficult to detect bacterial solutions with low concentrations; the color change unit method and the total cell nitrogen or total carbon method cannot specifically detect the concentration coefficient of a single bacteria in a mixed bacterial solution. These traditional microbial detection technologies can only meet the preliminary estimation of the total amount of bacteria, and it is difficult to give specific bacterial concentration data, and the detection of the concentration of the bacteria to be measured is extremely susceptible to interference from other bacteria. Therefore, when conducting quantitative analysis of the concentration of a single bacteria, it is imperative to provide a microbial detection technology with high precision, easy operation, strong anti-interference ability, high specificity, and a wide detection range.

[0003] Optical biosensors have recently been recognized as an attractive alternative to existing traditional pathogen detection methods. Optical biosensors can sense the interaction of microorganisms with analytes and correlate the observed optical signals with the concentration of microbial populations based on the measurement of photons involved in the process. Optical detection mainly involves measuring luminescence, fluorescence emission or absorbance. Adenosine triphosphate (ATP) bioluminescence is considered to be a very effective biosensor and is the main bioenergy source present in various microorganisms, reflecting the presence of living microorganisms. Sensitive, non-destructive and real-time detection can be performed.

[0004] The solution of luciferin and some luciferase will not react to produce bioluminescence in an environment without the support of energy substance triphosphoglycerate (ATP), but after encountering ATP, the bond will be broken to release a phosphate group and provide energy for the luciferase reaction of luciferin. Luciferin will release light after being degraded by luciferase, which is the basic principle of bioluminescence detection.

[0005] Today's food testing industry has broad prospects, but the existing technology faces problems such as low sensitivity, time-consuming, cumbersome steps, low accuracy, susceptibility to interference, narrow detection range, and low precision. Therefore, it is urgent to develop a bacteriophage bioluminescent array bacterial detection chip for the detection of microbial content in food with high sensitivity, high accuracy, strong specificity, short test time, simple operation, low cost, and wide detection range. Summary of the invention

[0006] The purpose of the present invention is to provide a bacteriophage bioluminescent array bacteria detection chip and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a bacteriophage bioluminescent array bacteria detection chip, which is characterized by comprising the following steps:

[0009] (1) adding a mixture of polydiallyldimethylammonium chloride, polylactic acid and LB culture medium into a chip mold and performing a curing treatment to obtain a chip substrate with a plurality of reaction pools;

[0010] (2) attaching and fixing the chip substrate to a plexiglass base to obtain a chip to be modified;

[0011] (3) performing bacteria modification, phage modification and luminescence modification in sequence in the reaction pool of the chip to be modified, and finally obtaining a phage bioluminescence array bacteria detection chip.

[0012] Preferably, the length, width and height of the chip substrate are 30-150 mm, 20-100 mm and 5-25 mm respectively.

[0013] Preferably, the reaction pool is hemispherical and has a diameter of 6 to 30 mm.

[0014] Preferably, the mass ratio of polydiallyldimethylammonium chloride, polylactic acid and LB culture medium in the mixed solution is 0.5-1.5:50-150:5-15.

[0015] Preferably, after the mixed solution is added to the chip mold, it is first allowed to stand at 15-35° C. for 3-4 hours and then subjected to a curing treatment, wherein the curing treatment temperature is 55-65° C. and the time is 1-2 hours.

[0016] Preferably, before the lamination process, the coated chip substrate and the organic glass base are firstly subjected to plasma treatment.

[0017] Preferably, the temperature of the plasma treatment is 15-35° C., and the time is 0.5-1.5 min.

[0018] Preferably, the temperature of the bonding and fixing is 75 to 85° C., and the time is 1 to 3 hours.

[0019] Preferably, the bacterial modification is to add 0.5 mL / well of bacterial solution into the reaction pool and allow to stand for modification.

[0020] Preferably, the bacterial solution is an LB solution of bacteria, and the concentration of the bacterial solution has an OD value of 1.0 to 2.0 at a light wavelength of 580 to 660 nm.

[0021] Preferably, the bacteria include one of Staphylococcus aureus, Escherichia coli, nitrifying bacteria, Clostridium tetani, lactic acid bacteria, Mycobacterium tuberculosis, Salmonella, Vibrio parahaemolyticus, Escherichia coli, Vibrio cholerae, Listeria monocytogenes, Brucella and Shigella.

[0022] Preferably, the bacterial modification time is 1 to 5 hours.

[0023] Preferably, the phage modification is to fill the reaction pool with the phage solution.

[0024] Preferably, the phage solution is prepared by using a DPBS buffer solution containing magnesium ions and calcium ions, and the concentration of the phage solution is 10 4 ~10 7 PFU / mL.

[0025] Preferably, the temperature of the phage modification is 15-35° C. and the time is 20-50 min.

[0026] Preferably, the phage modification also includes a phage proliferation process, and the phage proliferation temperature is 15 to 35° C. and the time is 8 to 12 hours.

[0027] Preferably, the luminescent modification is to fill the reaction pool with a luminescent reagent.

[0028] Preferably, the luminescent reagent is a mixture of luciferin, luciferase and PBS buffer in a mass volume ratio of 8-12 mg:0.08-0.12 mg:8-12 mL, the concentration of the PBS buffer is 8-12 mmol / L, and the pH value is 7.2-7.6.

[0029] Preferably, the single time of the luminescence modification is 1 to 1.5 hours, and the number of the luminescence modification is 3 to 5 times.

[0030] Preferably, the interval time of each luminescence modification is 10 to 20 minutes.

[0031] The present invention also provides a bacteriophage bioluminescent array bacteria detection chip obtained by the above preparation method.

[0032] The present invention also provides an application of the chip obtained by the above preparation method in preparing a product for detecting the content of pathogenic strains in food.

[0033] The present invention uses polydiallyldimethylammonium chloride, polylactic acid and LB culture medium to prepare a phage bioluminescent array bacteria detection chip substrate, and sequentially performs bacteria modification, phage modification and luminescence modification to obtain a phage bioluminescent array bacteria detection chip. The LB culture medium attracts bacteria to grow on the surface of a reaction pool; the high molecular weight substance polydiallyldimethylammonium chloride with positively charged groups on the surface firmly adsorbs the phage with negatively charged heads on the surface of the reaction pool.

[0034] The present invention captures and kills the bacteria to be tested by bacteriophage-specificity, causing the bacterial cell wall to dissociate and release ATP to supply energy to luciferase, which then decomposes luciferin and emits bright light.

[0035] The chip prepared by the present invention has high sensitivity, high accuracy and strong specificity, and the drug sensitivity test time for toxic strains is shortened to within 2 hours. In addition, the preparation process is simple, no large-scale equipment is required, the reaction conditions are mild, the cost is low and the safety factor is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The AI ​​design drawing of the chip mold in Example 1;

[0037] Figure 2 A top view of the phage bioluminescent array bacteria detection chip substrate prepared in Example 1;

[0038] Figure 3 A top view of the phage bioluminescent array bacterial detection chip substrate prepared in Example 1 (with Figure 2 Different structural dimensions are marked);

[0039] Figure 4 This is a front view of the bacteriophage bioluminescent array bacteria detection chip substrate prepared in Example 1;

[0040] Figure 5 This is a left view of the bacteriophage bioluminescent array bacteria detection chip substrate prepared in Example 1;

[0041] Figure 6 This is a schematic diagram of the fixing of the substrate of the bacteriophage bioluminescent array bacteria detection chip obtained in Example 1 to the base;

[0042] Figure 7 This is the linear regression line obtained after the phage bioluminescent array bacterial detection chip in Example 2 detects a series of Staphylococcus aureus strains with different concentration gradients. DETAILED DESCRIPTION

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] Example 1

[0045] Preparation of bacteriophage bioluminescent array bacterial detection chip

[0046] Preparation of bacteriophage bioluminescent array bacterial detection chip using Staphylococcus aureus as an example

[0047] (1) Preparation of phage bioluminescent array bacterial detection chip substrate:

[0048] Chip mold preparation: AI graphics are used to create designed graphics (such as Figure 1 As shown in the figure, the jdpaint software was imported, and the engraving path and depth were set as follows: the length, width and height of the substrate were 60 mm, 40 mm and 10 mm respectively; the perimeter of the hemispherical reaction pool was 12 mm, and the chip mold was obtained by engraving with an engraving machine;

[0049] Hydrophobic treatment of the chip mold: the chip mold is placed in a toluene solution of octadecyltrichlorosilane with a mass concentration of 2% for hydrophobic treatment for 30 minutes, and after the treatment, it is washed with a toluene solution for 3 times and dried at room temperature for 2 hours to obtain a hydrophobic chip mold;

[0050] Preparation of the phage bioluminescent array bacterial detection chip substrate: Pour a mixture of polydiallyldimethylammonium chloride, polylactic acid and LB culture medium in a mass ratio of 1:100:10 into a hydrophobic chip mold, let it stand at 25°C for 3.5 hours, and then dry it at 60°C for 1 hour to obtain the phage bioluminescent array bacterial detection chip substrate. The top view, front view and left view of the chip substrate are shown in the figure. Figures 2 to 5 shown.

[0051] LB medium: 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl were weighed and dissolved in 950 mL of distilled water, and the pH value was adjusted to 7.0 with 1 M NaOH, and the volume was fixed to 1 L. Autoclave at 121°C for 20 min to obtain LB solution, which was stored at 4°C.

[0052] The phage bioluminescent array bacterial detection chip substrate and the organic glass plate with a length, width and height of 60 mm, 40 mm and 20 mm respectively were treated with a plasma machine at 25°C for 1 min. Figure 6 The position relationship shown is placed at 80°C for 2 hours to obtain the chip to be modified.

[0053] (2) Preparation of phage bioluminescent array bacterial detection chip:

[0054] Bacterial modification:

[0055] Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl and dissolve them in 950 mL of distilled water. Adjust the pH to 7.0 with 1 M NaOH and make the volume 1 L. Autoclave at 121°C for 20 min to obtain LB solution, which is then stored at 4°C.

[0056] Weigh 8.2g NaCl, 0.2g KCl, 2.9g Na 2 HPO 4 , 0.2g KH 2 PO 4 and 0.5 mL of Tween 20, add distilled water to make up to 1000 mL to obtain 0.01 M PBST buffer.

[0057] Staphylococcus aureus was mixed with LB solution to prepare a Staphylococcus aureus solution with an OD of 1.6 at a wavelength of 600 nm.

[0058] 2 mL of Staphylococcus aureus solution was added to the reaction pools for modification for 3 h. After the bacteria were adsorbed into the reaction pools, they were washed three times with 0.01 M PBST buffer at pH 7.4 and dried at 25° C. until no liquid remained in the reaction pools.

[0059] Phage modification:

[0060] Weigh 0.2 g potassium chloride, 0.2 g monopotassium phosphate, 8 g sodium chloride, 2.1 g disodium hydrogen phosphate heptahydrate, 0.1 g calcium chloride, and 0.1 g magnesium chloride hexahydrate, and dilute to 1 L with secondary deionized water to obtain a DPBS buffer diluent containing calcium and magnesium ions. Mix it with the bacteriophage to a concentration of 10 6 PFU / mL phage solution.

[0061] Fill the reaction pool after bacteria modification with phage solution, modify at 25℃ for 30min. After modification, proliferate at 25℃ for 10h. After proliferation, wash three times with 0.01M PBST buffer at pH=7.4, and dry at 25℃ until no liquid remains in the reaction pool.

[0062] Glow Modification:

[0063] Weigh 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 , 0.24g KH 2 PO 4 Dissolve in 800 mL of distilled water, adjust the solution to 7.4 with HCl, and finally add distilled water to make up to 1 L to obtain 0.01 M PBS buffer.

[0064] 5 g of luciferin, 2 g of luciferase and 100 mL of PBS buffer were mixed evenly to obtain a mixed solution of luciferin and luciferase.

[0065] The reaction pool was filled with the mixed solution of luciferin and luciferase for modification 4 times, each time for 1 hour, and each modification interval was 15 minutes, so as to obtain a bacteriophage bioluminescent array bacterial detection chip.

[0066] Example 2

[0067] Detection of microbial content in food using Staphylococcus aureus as an example

[0068] (1) The concentration is 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 The standard solution of Staphylococcus aureus with a CFU / mL is prepared by using a sterile physiological saline with a mass concentration of 0.90%.

[0069] (2) Use a syringe to draw 100 μL of each of the 8 independent units of the sterilized chip prepared in Example 1 (7 groups in total) and 1 group of sterile saline with a mass concentration of 0.90% into the corresponding reaction pool positions, respectively, and culture at 37°C for 3 hours, with 3 repetitions.

[0070] (3) The reaction pool of the prepared phage bioluminescent array bacterial detection chip was immersed in a PBST buffer solution with a pH value of 7.4 for 2 hours; and dried in a sterile room for 1 hour until no liquid remained in the reaction pool.

[0071] (4) The bioluminescent array bacterial detection chip enriched with Staphylococcus aureus strains in step (3) was placed in a bioluminescent detector, and the relative light unit values ​​in the eight reaction pools were recorded. The relative light unit value of the group injected with sterile saline at a mass concentration of 0.90% was recorded as (BL 0 ), and then the relative light unit values ​​in the remaining 7 groups of reaction pools are recorded in order as (BL i ), BL i With BL 0 Subtract and obtain the relative light unit value (ΔBL i ). That is, the relative light unit values ​​of different concentrations of Staphylococcus aureus strains are BL 2 BL 3 BL 4 BL 5 BL 6BL 7 BL 8 According to the relative light unit values ​​of the above-mentioned different concentrations of Staphylococcus aureus strains and the Log10 logarithm of the concentration, a linear fitting was performed, and the concentration of the Staphylococcus aureus strain was used as the horizontal axis and the relative light unit value of the Staphylococcus aureus strain was used as the vertical axis to draw an image, and a linear regression equation between the concentration and the relative light unit value was obtained, as shown in FIG. Figure 7 As shown. Record the linear regression equation parameters as the reference equation.

[0072] (5) One apple (278 g), one banana (156 g), 100 g prune, 100 g mushroom, 100 g cowpea, 100 g leek, 100 g celery, and 100 g Chinese cabbage were purchased randomly from the market and divided into 8 groups. Each group of fruits and vegetables was soaked in 100 mL of sterile 0.90% saline for 1 h.

[0073] (6) Use a syringe to draw 100 μL of the saline solution used to soak fruits and vegetables in step (5) from the 8 independent units of a new sterilized chip, inject it into the reaction pool, and incubate at 37° C. for 3 h.

[0074] (7) The reaction pool of the prepared phage bioluminescent array bacterial detection chip was immersed in a PBST buffer solution with a pH value of 7.4 for 2 hours; and dried in a sterile room for 1 hour, and no liquid remained in the reaction pool.

[0075] (8) Place the bioluminescent array bacterial detection chip after drying in step (7) in a bioluminescent detector, record the relative light unit values ​​in the 8 groups of reaction pools, and record the relative light unit values ​​in the 8 groups of reaction pools in order as (BL i '), BL i 'With BL 0 Subtract and obtain the corresponding relative light unit value (ΔBL i '). That is, the relative light unit values ​​of Staphylococcus aureus strains in different samples are BL 1 ', BL 2 ', BL 3 ', BL 4 ', BL 5 ', BL 6 ', BL 7 ', BL 8 ', find the logarithmic value of the concentration corresponding to the relative light unit value. Then add 3 to the logarithmic value of the concentration of Staphylococcus aureus found and take the exponent 10 to obtain the concentration value of Staphylococcus aureus on different varieties of fruits and vegetables.

[0076] The test results showed that the concentration of Staphylococcus aureus was 56 CFU / g in apples, 106 CFU / g in bananas, 284 CFU / g in prunes, 51 CFU / g in shiitake mushrooms, 47 CFU / g in cowpeas, 46 CFU / g in leeks, and no Staphylococcus aureus was detected in celery and Chinese cabbage.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a bacteriophage bioluminescent array bacterial detection chip, characterized in that: The steps include: (1) adding a mixture of polydiallyldimethylammonium chloride, polylactic acid and LB culture medium into a chip mold and performing a curing treatment to obtain a chip substrate with a plurality of reaction pools; (2) attaching and fixing the chip substrate to a plexiglass base to obtain a chip to be modified; (3) sequentially performing bacteria modification, phage modification, and luminescence modification in the reaction pool of the chip to be modified, and finally obtaining a phage bioluminescence array bacteria detection chip; The bacteria used in the bacterial modification include one of Staphylococcus aureus, Escherichia coli, nitrifying bacteria, Clostridium tetani, lactic acid bacteria, Mycobacterium tuberculosis, Salmonella, Vibrio parahaemolyticus, Escherichia coli, Vibrio cholerae, Listeria monocytogenes, Brucella and Shigella dysenteriae; The luminescent modification is to fill the reaction pool with the luminescent reagent; The luminescent reagent is a mixed solution of luciferin, luciferase and PBS buffer in a mass volume ratio of 8-12 mg: 0.08-0.12 mg: 8-12 mL.

2. The preparation method according to claim 1, characterized in that: The length, width and height of the chip substrate are respectively 30-150 mm, 20-100 mm and 5-25 mm; the reaction pool is hemispherical with a diameter of 6-30 mm.

3. The preparation method according to claim 2, characterized in that: The mass ratio of polydiallyldimethylammonium chloride, polylactic acid and LB culture medium in the mixed solution is 0.5-1.5:50-150:5-15; After the mixed solution is added into the chip mold, it is first allowed to stand at 15-35° C. for 3-4 hours, and then subjected to a curing treatment, wherein the curing treatment temperature is 55-65° C. and the curing time is 1-2 hours.

4. The preparation method according to claim 3, characterized in that: Before the bonding process, the coated chip substrate and the organic glass base are firstly subjected to plasma treatment; The temperature of the plasma treatment is 15-35°C and the time is 0.5-1.5min; The temperature of the bonding and fixing is 75 to 85° C., and the time is 1 to 3 hours.

5. The preparation method according to claim 4, characterized in that: The bacterial modification is to add 0.5 mL / well of bacterial solution into the reaction pool and leave it to stand for modification; The bacterial solution is an LB solution of bacteria, and the concentration of the bacterial solution has an OD value of 1.0 to 2.0 at a light wavelength of 580 to 660 nm; the bacterial modification time is 1 to 5 hours.

6. The preparation method according to claim 5, characterized in that: The phage modification is to fill the reaction pool with the phage solution; The phage solution is prepared by using a DPBS buffer solution containing magnesium ions and calcium ions. The concentration of the phage solution is 10 4 ~10 7 PFU / mL; The phage modification temperature is 15-35°C and the time is 20-50 minutes; The phage modification also includes the process of phage proliferation, and the temperature of the phage proliferation is 15-35° C. and the time is 8-12 hours.

7. The preparation method according to claim 6, characterized in that: The concentration of the PBS buffer used in the luminescent modification in step (3) is 8 to 12 mmol / L, and the pH value is 7.2 to 7.6; The single modification time of the luminescence modification is 1 to 1.5 hours, the number of the luminescence modification is 3 to 5 times, and the interval time of each luminescence modification is 10 to 20 minutes.

8. The bacteriophage bioluminescent array bacteria detection chip obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the chip according to claim 8 in preparing a product for detecting the content of pathogenic strains in food.

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