CN110220951B - Method for detecting salmonella typhimurium in food by using integrated device - Google Patents
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Abstract
The patent discloses a method for detecting salmonella typhimurium in food by using an integrated device, and relates to the field of nucleic acid detection. The integrated device is a multi-layer plate-shaped structure consisting of a magnetic plate, a paper electrode chip and a pasting film, realizes the continuous introduction of a sample, a purification buffer solution and an amplification reagent by sliding the middle magnetic plate, and an FTA card embedded in the magnetic plate is used for extracting nucleic acid from bacteria and is used as a reaction chamber for subsequent isothermal reaction. The method utilizes the integrated device to complete the nucleic acid detection steps of nucleic acid extraction, isothermal amplification and electrochemical detection of the salmonella typhimurium, avoids the complicated operation steps and the requirements on precise instruments of the traditional method, quickly and sensitively realizes the detection of the salmonella typhimurium in egg white, drinking water and milk samples, has the analysis performances of quickness, convenience, low cost, high efficiency, sensitivity and the like, and has potential application value for food safety analysis.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a method for detecting salmonella typhimurium in food by using an integrated device to complete the steps of nucleic acid extraction, isothermal amplification and detection.
Background
In recent years, diseases caused by microorganisms continuously burst around the world, wherein many diseases are highly contagious, highly harmful, difficult to diagnose and treat, and cause serious casualties and economic losses worldwide, thus causing serious threats to the life safety and production life of people. Salmonella typhimurium is a non-adaptive or pantropic Salmonella, has a wide range of hosts, and is one of the most highly infected bacterial types in all countries in the world at present. The bacteria can cause various infectious diseases of poultry and mammals, and can also cause human infection, and have important public health significance.
Salmonella typhimurium is widely distributed in the natural world and is present in the intestinal tract, muscles and internal organs of various animals such as poultry, livestock and rodents. The strain has strong resistance in external environment, can rapidly propagate at normal temperature, and can pollute food and drinking water. In recent years, food poisoning by salmonella typhimurium has also been higher than that of the first few cases of bacterial food poisoning. Therefore, rapid and sensitive detection of salmonella typhimurium in food is becoming increasingly important.
At present, the detection of salmonella typhimurium requires enrichment culture, separation identification, biochemical identification, serological identification and other procedures. The whole process takes too long time, and the optimal period of treatment of the patient can be delayed. In order to solve this problem, it is necessary to develop a method for rapidly and sensitively detecting Salmonella typhimurium from food.
Disclosure of Invention
The invention aims to solve the technical problem of constructing a method capable of sensitively and conveniently detecting salmonella typhimurium in food.
A method for detecting Salmonella typhimurium in food by using an integrated device is characterized by comprising the following steps:
(1) design and manufacture of devices
The integrated detection device is designed into a multilayer plate-shaped style, and the core structure is divided into an upper layer sample plate, a middle layer sliding plate and a lower layer electrode plate which are constructed by combining a magnetic plate with a sticking film and a paper electrode chip; the upper plate is provided with three round holes with the diameter of 5 mm and a square groove with the depth of 2 mm and the length of 3 cm, the three round holes are respectively used for dripping food samples, purification buffer solution and amplification reagents, the square groove is used for placing paper chips printed with carbon electrodes and silver electrodes, the middle layer sliding plate is composed of a slidable magnetic plate and two fixed magnetic stripes, the slidable magnetic plate is provided with a round hole with the diameter of 5 mm, an FTA disc (used for extracting nucleic acid from the food samples) with the diameter of 5 mm is plugged in the round hole, the FTA disc is aligned with the round hole of the upper plate by drawing the slidable magnetic plate, the dripping of biological samples, purification buffer solution and amplification reagents can be completed through the round hole of the upper layer, the lower plate comprises an absorption plate with the size of 10 cm multiplied by 5 cm and a magnetic plate with the size of 5 cm multiplied by 5 cm, the square groove with the depth of 2 mm and the length of 3, the absorption plate is used for absorbing the waste after cell lysis from the FTA disc, and the square groove is used for placing the paper chip printed with the working electrode; a closed amplification reaction chamber is constructed by utilizing the attraction of the magnetic plate and the adhesive film so as to ensure that isothermal amplification reaction can be smoothly carried out in the reaction chamber; the manufacturing steps of the device are as follows: cutting the magnetic plate and the adhesive film into 15 cm multiplied by 5 cm and the Whatman paper into 10 cm multiplied by 5 cm by a digital cutting machine, and punching a round hole with the diameter of 5 mm at a specific position of the magnetic plate and the adhesive film by a punching machine; the specific steps of the construction of the paper electrode chip are as follows: printing patterns on chromatographic paper by using a wax printing technology, placing the patterns of the printed wax at a constant temperature of 150 ℃ for 2 min, wherein the position where the wax is not printed is a hydrophilic area, the shape of a hydrophobic barrier of a paper electrode chip is designed by Adobe illustrator CS6 software, and the patterns of the Au nano particles are formed by printing a silver/silver chloride reference electrode, a carbon counter electrode and a carbon working electrode by using silk screen, and in addition, the Au nano particles are modified in the working area of the paper electrode chip by an auxiliary area (the diameter is 7 mm) and a working area (the diameter is 7 mm), wherein the specific steps of modifying the Au nano particles in the working area of the paper electrode chip are as follows: heating 80 mL of distilled water to 90 ℃, adding 0.8 mL of chloroauric acid solution with the mass fraction of 1%, continuing to heat to 96 ℃ for 1 minute, finally adding 2.8 mL of sodium citrate with the mass fraction of 1%, heating for 8 minutes to obtain a gold seed solution, dropwise adding 20 mu L of the gold seed solution into a hydrophilic area of a working layer, standing, airing, repeating for three times, and preparing the working electrode modified with gold nanoparticles; stacking the magnetic plate, the paper electrode chip and the adhesive film in sequence, and performing extrusion forming;
(2) salmonella typhimurium culture
The culture steps of the salmonella typhimurium are as follows: salmonella typhimurium was cultured overnight at 37 ℃ on Luria-Bertani (LB) plates, and then a single Salmonella typhimurium colony was picked to puncture and grown overnight at 37 ℃ in 10 ml LB liquid medium while shaking at 110 rpm; measuring the absorbance (OD 600) of the bacterial suspension at 600 nm with an ultraviolet spectrophotometer to determine the concentration of the bacteria;
(3) detection of bacteria in food using an integrated device
Diluting the bacterial suspension in the step (2) by 1, 10, 100, 500 and 1000 times respectively by using PBS (pH 7.4), adding equivalent diluent into a food sample respectively, and mixing uniformly;
the device mainly realizes the addition of food samples, purification reagents and amplification reagents by sliding the middle magnetic plate, and the FTA disc is aligned with the sample hole of the upper plate by sliding the middle magnetic plate; dropwise adding a food sample added with the bacterial diluent in equal quantity onto an FTA disc; drying at room temperature for 15 min for lysing bacteria and extracting nucleic acid; after 15 min, sliding the middle magnetic plate to align the FTA disc with the cleaning hole of the upper plate, dropwise adding a purification buffer solution, and cleaning bacteria cracking waste; heating at 65 deg.C for 5 min, and drying FTA disc; after drying, continuously sliding the middle magnetic plate to align the FTA disc with the reagent hole of the upper plate, and dropwise adding the isothermal amplification reaction mixed solution; sliding the middle magnetic plate again until the white mark line is exposed, and sliding the FTA disc to the closed amplification reaction chamber; the whole device is placed on a hot plate and heated at 65 ℃ for 60 min, so that isothermal amplification reaction is fully carried out; after 60 min, the device was removed from the hot plate, after cooling to room temperature, the carbon, silver and gold electrodes of the device were connected to an electrochemical workstation, and the current response was measured and recorded with Differential Pulse Voltammetry (DPV); the measurement parameters were as follows: the potential range is-0.3 to-0.1V, the modulation amplitude is 0.05V, the pulse width is 0.05 s, and the sample width is 0.0167 s;
(4) bacterial detection line assay in food
Changing the concentration of the bacteria in the food sample in the step (3) into a series of concentration gradients, then carrying out electrochemical detection under the same parameter setting, and obtaining a working curve through an electrochemical signal scanning curve of the bacteria in the food sample under different concentration gradients so as to realize quantitative analysis.
The invention has the advantages of
(1) The detection of the salmonella typhimurium in food is sensitively and conveniently realized by utilizing the integrated device;
(2) compared with the traditional bacteria monitoring method, the method avoids complicated operation steps and time-consuming culture process;
(3) the monitoring mode of 'sample-answer' can be realized in the environment with limited resources.
Drawings
FIG. 1 is an experimental schematic of the method described herein, and FIG. 2 is an operational diagram of the method described herein.
Detailed Description
For a better understanding of the invention, the following further illustrates the invention with reference to examples and drawings, but the invention is not limited to the following embodiments.
Example 1
A method for detecting Salmonella typhimurium in food by using an integrated device is characterized by comprising the following steps:
(1) design and manufacture of devices
The integrated detection device is designed into a multilayer plate-shaped style, and the core structure is divided into an upper layer sample plate, a middle layer sliding plate and a lower layer electrode plate which are constructed by combining a magnetic plate with a sticking film and a paper electrode chip; the upper plate is provided with three round holes with the diameter of 5 mm and a square groove with the depth of 2 mm and the length of 3 cm, the three round holes are respectively used for dripping food samples, purification buffer solution and amplification reagents, the square groove is used for placing paper chips printed with carbon electrodes and silver electrodes, the middle sliding plate is composed of a slidable magnetic plate and two fixed magnetic stripes, the slidable magnetic plate is provided with a round hole with the diameter of 5 mm, an FTA disc (used for extracting nucleic acid from the food samples) with the diameter of 5 mm is plugged in the round hole, the FTA disc is aligned with the round hole of the upper plate by drawing the slidable magnetic plate, the dripping of the food samples, the purification buffer solution and the amplification reagents can be completed through the round hole of the upper layer, the lower plate comprises an absorption plate with the size of 10 cm multiplied by 5 cm and a magnetic plate with the size of 5 cm multiplied by 5 cm, and the square groove with the depth of 2 mm and, the absorption plate is used for absorbing the waste after cell lysis from the FTA disc, and the square groove is used for placing the paper chip printed with the working electrode; a closed amplification reaction chamber is constructed by utilizing the attraction of the magnetic plate and the adhesive film so as to ensure that isothermal amplification reaction can be smoothly carried out in the reaction chamber; the manufacturing steps of the device are as follows: cutting the magnetic plate and the adhesive film into 15 cm multiplied by 5 cm by a digital cutting machine, cutting the Whatman paper into 10 cm multiplied by 5 cm, and punching a round hole with the diameter of 5 mm at a specific position of the magnetic plate and the adhesive film by a punching machine; the specific steps of the construction of the paper electrode chip are as follows: printing patterns on chromatographic paper by using a wax printing technology, placing the patterns of the printed wax at a constant temperature of 150 ℃ for 2 min, wherein the position where the wax is not printed is a hydrophilic area, the shape of a hydrophobic barrier of a paper electrode chip is designed by Adobe illustrator CS6 software, and the patterns of the Au nano particles are formed by printing a silver/silver chloride reference electrode, a carbon counter electrode and a carbon working electrode by using silk screen, and in addition, the Au nano particles are modified in the working area of the paper electrode chip by an auxiliary area (the diameter is 7 mm) and a working area (the diameter is 7 mm), wherein the specific steps of modifying the Au nano particles in the working area of the paper electrode chip are as follows: heating 80 mL of distilled water to 90 ℃, adding 0.8 mL of chloroauric acid solution with the mass fraction of 1%, continuing to heat to 96 ℃ for 1 minute, finally adding 2.8 mL of sodium citrate with the mass fraction of 1%, heating for 8 minutes to obtain a gold seed solution, dropwise adding 20 mu L of the gold seed solution into a hydrophilic area of a working layer, standing, airing, repeating for three times, and preparing the working electrode modified with gold nanoparticles; stacking the magnetic plate, the paper electrode chip and the adhesive film in sequence, and performing extrusion forming;
(2) salmonella typhimurium culture
The culture steps of the salmonella typhimurium are as follows: salmonella typhimurium was cultured overnight at 37 ℃ on Luria-Bertani (LB) plates, and then a single Salmonella typhimurium colony was picked to puncture and grown overnight at 37 ℃ in 10 ml LB liquid medium while shaking at 110 rpm; measuring the absorbance (OD 600) of the bacterial suspension at 600 nm with an ultraviolet spectrophotometer to determine the concentration of the bacteria;
(3) detection of Salmonella typhimurium in food using an integrated device
Diluting the bacterial suspension in the step (2) by 1, 10, 100, 500 and 1000 times respectively by using PBS (pH 7.4), adding 10 mu L of diluent into 90 mu L of egg white respectively, and mixing uniformly; the device mainly realizes the addition of food samples, purification reagents and amplification reagents by sliding the middle magnetic plate, and the FTA disc is aligned with the sample hole of the upper plate by sliding the middle magnetic plate; taking 10 mu L of egg white added with salmonella typhimurium, and dropwise adding the egg white to the FTA disc; drying at room temperature for 15 min for cracking Salmonella typhimurium and extracting nucleic acid; after 15 min, sliding the middle magnetic plate to align the FTA disc with the cleaning hole of the upper plate, dropwise adding a purification buffer solution, and cleaning the salmonella lysis waste; heating at 65 deg.C for 5 min, and drying FTA disc; after drying, continuously sliding the middle magnetic plate to align the FTA disc with the reagent hole of the upper plate, and dropwise adding the isothermal amplification reaction mixed solution; sliding the middle magnetic plate again until the white mark line is exposed, and sliding the FTA disc to the closed amplification reaction chamber; the whole device is placed on a hot plate and heated at 65 ℃ for 60 min, so that isothermal amplification reaction is fully carried out; after 60 min, the device was removed from the hot plate, after cooling to room temperature, the carbon, silver and gold electrodes of the device were connected to an electrochemical workstation, and the current response was measured and recorded with Differential Pulse Voltammetry (DPV); the measurement parameters were as follows: the potential range is-0.3 to-0.1V, the modulation amplitude is 0.05V, the pulse width is 0.05 s, and the sample width is 0.0167 s;
(4) salmonella typhimurium detection line assay in food
Changing the concentration of the salmonella typhimurium in the food sample in the step (3) into a series of concentration gradients, then carrying out electrochemical detection under the same parameter setting, and obtaining a working curve through electrochemical signal scanning curves of the salmonella typhimurium in the egg white under different concentration gradients so as to realize quantitative analysis.
Example 2
The detection procedure was the same as in example 1, except that: and (4) adding the salmonella typhimurium diluent into the food sample obtained in the step (3) to obtain drinking water.
Example 3
The detection procedure was the same as in example 1, except that: and (4) adding the salmonella typhimurium diluent into the food sample obtained in the step (3) to obtain the milk.
Sequence listing
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Claims (3)
1. A method for detecting Salmonella typhimurium in food by using an integrated device is characterized by comprising the following steps:
(1) design and manufacture of devices
The integrated detection device is designed into a multilayer plate shape, the core structure is divided into an upper layer sample plate, a middle layer sliding plate and a lower layer electrode plate, and the integrated detection device is constructed by combining a magnetic plate with a paper chip on which a film and a printed electrode are pasted; the upper layer sample plate is provided with three round holes with the diameter of 5 mm and a square groove with the depth of 2 mm and the length of 3 cm, the three round holes are respectively used for dripping a food sample, a purification buffer solution and an amplification reagent, and the square groove is used for placing a paper chip printed with a carbon electrode and a silver electrode; the middle sliding plate consists of a slidable magnetic plate and two fixed magnetic strips, the slidable magnetic plate is provided with a round hole with the diameter of 5 mm, an FTA disc with the diameter of 5 mm and used for extracting nucleic acid from a food sample is plugged in the round hole, the slidable magnetic plate is pulled to align the FTA disc with the round hole of the upper sample plate, and the food sample, the purification buffer solution and the amplification reagent can be dripped through the upper round hole; the lower electrode plate comprises an absorption plate with the size of 10 cm multiplied by 5 cm and a magnetic plate with the size of 5 cm multiplied by 5 cm, a square groove with the depth of 2 mm and the length of 3 cm is arranged on the magnetic plate, the absorption plate is used for absorbing waste after cell lysis from the FTA disc, and the square groove is used for placing a paper chip printed with a working electrode; a closed amplification reaction chamber is constructed by utilizing the attraction of the magnetic plate and the adhesive film so as to ensure that isothermal amplification reaction can be smoothly carried out in the reaction chamber; the manufacturing steps of the device are as follows: cutting the magnetic plate and the adhesive film into 15 cm multiplied by 5 cm by a digital cutting machine, cutting the Whatman paper into 10 cm multiplied by 5 cm, and punching a round hole with the diameter of 5 mm at a specific position of the magnetic plate and the adhesive film by a punching machine; the specific steps of the construction of the paper chip after the electrode printing are as follows: printing patterns on chromatographic paper by using a wax printing technology, placing the patterns of the printed wax at a constant temperature of 150 ℃ for 2 min, wherein the position where the wax is not printed is a hydrophilic area, the shape of a hydrophobic barrier of a paper chip after an electrode is printed is designed by Adobe illustrator CS6 software, a silver/silver chloride reference electrode, a carbon counter electrode and a carbon working electrode are printed by using silk screen printing, in addition, the method also comprises an auxiliary area with the diameter of 7mm and a working area with the diameter of 7mm, and the specific steps of modifying Au nano particles in the working area of the paper chip after the electrode is printed are as follows: heating 80 mL of distilled water to 90 ℃, adding 0.8 mL of chloroauric acid solution with the mass fraction of 1%, continuing to heat to 96 ℃ for 1 minute, finally adding 2.8 mL of sodium citrate with the mass fraction of 1%, heating for 8 minutes to obtain a gold seed solution, dropwise adding 20 mu L of the gold seed solution into a hydrophilic area of a working layer, standing, airing, repeating for three times, and preparing the working electrode modified with gold nanoparticles; stacking the magnetic plate, the paper chip printed with the electrode and the adhesive film in sequence, and performing extrusion forming;
(2) salmonella typhimurium culture
The culture steps of the salmonella typhimurium are as follows: salmonella typhimurium was cultured overnight at 37 ℃ on Luria-Bertani (LB) plates, and then a single Salmonella typhimurium colony was picked to puncture and grown overnight at 37 ℃ in 10 ml LB liquid medium while shaking at 110 rpm; measuring the absorbance (OD 600) of the bacterial suspension at 600 nm with an ultraviolet spectrophotometer to determine the concentration of the bacteria;
(3) monitoring Salmonella typhimurium in food using an integrated device
Diluting the bacterial suspension in the step (2) by 1, 10, 100, 500 and 1000 times respectively by using PBS buffer solution with pH 7.4, adding 10 mu L of diluent into 90 mu L of egg white respectively, and mixing uniformly; the device mainly realizes the addition of food samples, purification reagents and amplification reagents by sliding the middle magnetic plate, and the FTA disc is aligned with the sample hole of the upper sample plate by sliding the middle magnetic plate; taking 10 mu L of egg white added with salmonella typhimurium, and dropwise adding the egg white to the FTA disc; drying at room temperature for 15 min for cracking Salmonella typhimurium and extracting nucleic acid; after 15 min, sliding the middle magnetic plate to align the FTA disc with the cleaning holes of the upper sample plate, dropwise adding a purification buffer solution, and cleaning the salmonella lysis waste; heating at 65 deg.C for 5 min, and drying FTA disc; after drying, continuously sliding the middle magnetic plate to align the FTA disc with the reagent hole of the upper sample plate, and dropwise adding the isothermal amplification reaction mixed solution; sliding the middle magnetic plate again until the white mark line is exposed, and sliding the FTA disc to the closed amplification reaction chamber; the whole device is placed on a hot plate and heated at 65 ℃ for 60 min, so that isothermal amplification reaction is fully carried out; after 60 min, the device was removed from the hot plate, after cooling to room temperature, the carbon, silver and gold electrodes of the device were connected to an electrochemical workstation, and the current response was measured and recorded with Differential Pulse Voltammetry (DPV); the measurement parameters were as follows: the potential range is-0.3 to-0.1V, the modulation amplitude is 0.05V, the pulse width is 0.05 s, and the sample width is 0.0167 s;
(4) salmonella typhimurium detection line assay in food
Changing the concentration of the salmonella typhimurium in the food sample in the step (3) into a series of concentration gradients, then carrying out electrochemical detection under the same parameter setting, and obtaining a working curve through electrochemical signal scanning curves of the salmonella typhimurium in the egg white under different concentration gradients so as to realize quantitative analysis.
2. The method of claim 1, wherein the sample of food in step (3) is drinking water.
3. The method of claim 1, wherein the food sample in step (3) is milk.
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