CN116026815A

CN116026815A - Halal meat food detection device and detection method thereof

Info

Publication number: CN116026815A
Application number: CN202210867268.XA
Authority: CN
Inventors: 程楠; 徐瑗聪; 张倩; 李昀怡; 庞晓旭; 黄昆仑; 罗云波
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-07-21
Filing date: 2022-07-21
Publication date: 2023-04-28
Anticipated expiration: 2042-07-21
Also published as: CN116026815B

Abstract

The invention relates to a halal meat food detection device and a detection method thereof, wherein the halal meat food detection device comprises: the detection assembly comprises a sample tube, a fixed column, a capillary tube and a test paper board, wherein the fixed column is detachably covered on the sample tube so as to be capable of opening or closing the sample tube in an operable manner; the test paper board is operably positioned on the fixed column or falls into the sample tube from the fixed column; the capillary is wound on the fixed column, one end of the capillary is positioned outside the sample tube, and the other end of the capillary extends into the sample tube so as to be capable of operationally injecting the liquid in the capillary into the sample tube; the heater is provided with a heating cavity, and at least the bottom of the sample tube can extend into the heating cavity.

Description

Halal meat food detection device and detection method thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a halal meat food detection device and a detection method thereof.

Background

Islamic is a banned swine-derived food product, and some Islamic countries have established strict regulations that require manufacturers and importers to cover their products with Islamic certificates to distinguish them from non-Islamic products. However, the adulteration of meat products frequently occurs, and some manufacturers are able to blend pork into halal meat products in order to reduce the cost of pork, which is lower than beef and mutton. After the meat product is added with seasonings, essence and spices and processed for a plurality of times, meat components cannot be completely distinguished only through sense organs, if pork components are mixed into halal foods, religious beliefs of Islamic churn are greatly violated, and religious contradiction is even caused.

Disclosure of Invention

In view of the above analysis, the present invention provides a halal meat detection device and a halal meat detection method for solving the above problems.

In one aspect, the present invention provides a halal meat product detection device comprising:

the detection assembly comprises a sample tube, a fixed column, a capillary tube and a test paper board, wherein the fixed column is detachably covered on the sample tube so as to be capable of opening or closing the sample tube in an operable manner; the test paper board is operably positioned on the fixed column or falls into the sample tube from the fixed column; the capillary is wound on the fixed column, one end of the capillary is positioned outside the sample tube, and the other end of the capillary extends into the sample tube so as to be capable of operationally injecting the liquid in the capillary into the sample tube;

the heater is provided with a heating cavity, and at least the bottom of the sample tube can extend into the heating cavity.

Further, the fixing column is provided with a slot matched with the test paper board, and the slot penetrates through two ends of the fixing column;

two opposite side walls of the slot are respectively provided with a perforation for the contact pin to pass through;

when the test paper board is positioned on the fixing column, the pins are positioned in the two through holes, and the bottoms of the test paper boards are inserted into the slots and abutted against the pins so as to fix the test paper board in the slots; after the contact pin is taken out of the through hole, the test paper plate falls into the sample tube from the fixed column.

Further, the fixing column includes a column portion and a bottom plate portion, the column portion being located above the bottom plate portion;

the bottom plate part is matched with the sample tube, and the bottom plate part is detachably covered on the tube orifice of the sample tube;

the slot extends from the top end of the column part to the bottom end of the bottom plate part;

the through holes are positioned at the bottom of the side wall of the column body, and the connecting lines of the two through holes are perpendicularly intersected with the central line of the slot.

Further, the column part is provided with a clamping plate part, and the bottom plate part is provided with an inserting hole for the capillary to pass through;

the capillary tube is clamped into the clamping plate part and is wound in a downward rotating mode along the column part, and the tail end of the capillary tube extends into the sample tube from the jack.

Further, the bottom plate part is provided with a clamping groove part;

the mouth of pipe of sample tube be equipped with draw-in groove portion matched with annular protruding, annular protruding at least a portion can dismantle the embedding to draw-in groove portion.

Further, the halal meat food detection device further comprises an assembling plate detachably arranged on the heating cavity so as to be capable of opening or closing the heating cavity in an operable manner;

the assembly plate is provided with assembly parts with the same number as the detection assemblies, the detection assemblies are detachably arranged in the assembly parts, and the detection assemblies correspond to the assembly parts one by one.

Further, the bottom plate part of the fixed column is matched with the assembling part, and the bottom plate part is detachably arranged on the assembling part;

the assembly part is provided with a chute, and the bottom plate part is provided with a sliding block matched with the chute.

Further, the detecting component is provided with a plurality of capillaries, and at least one of the color, the pattern, the thickness and the number of the capillaries is uniquely corresponding to the capillaries so as to be used for distinguishing the types of the liquid in the capillaries.

Further, the heater is also provided with a storage cavity for storing the detection assembly.

On the other hand, the invention provides a halal meat food detection method, which is implemented by the halal meat food detection device and comprises the following steps:

step one: loading the RPA reagent, the CRISPR/Cas reagent and the running buffer into different capillaries respectively, and sealing by using Vaseline;

placing an extracted genome of a sample to be detected into a sample tube;

step two: winding the three capillaries loaded in the first step on a fixed column;

the fixed column is covered on the sample tube;

the tail ends of the three capillaries are respectively extended into the sample tube;

inserting the contact pins into the two through holes, and inserting the test paper board into the slot and abutting against the contact pins;

placing the sample tube into a heating cavity;

step three: heating the heating cavity to 37 ℃ by a heater, blowing the RPA reagent into the sample tube, and heating for 10min at 37 ℃; then, blowing CRISPR/Cas reagent into the sample tube, and continuously heating at 37 ℃ for 20min; blowing a running buffer into the sample tube, and finally pulling out the test paper board from the insertion pin in the perforation to drop into the sample tube;

step four: and judging whether pork is mixed in the sample to be detected by observing the color reaction of the test paper board.

In the first step, three capillaries are respectively filled with 2-20 mu L of RPA reagent, 20-150 mu L of CRISPR/Cas reagent and 80-200 mu L of running buffer reagent; preferably, 5-12. Mu.L of RPA reagent, 40-100. Mu.L of CRISPR/Cas reagent, 80-150. Mu.L of running buffer reagent, more preferably, 9.5. Mu.L of RPA reagent, 70. Mu.L of CRISPR/Cas reagent, 120. Mu.L of running buffer reagent are contained.

The three capillaries are different in color, namely the types of reagents in the capillaries are distinguished through the colors of the capillaries.

The RPA reagent comprises 2-10. Mu.L of resuspended RPA enzyme solution, 0.5-5. Mu.L of ddH ₂ O, 0.2-1.5. Mu.L of RPA-F (concentration: 10. Mu. Mol/L), 0.2-1.5. Mu.L of RPA-R (concentration: 10. Mu. Mol/L), and 0.2-1.5. Mu.L of magnesium acetate solution (concentration: 280 mmol/L); preferably, 5.9. Mu.L of resuspended RPA enzyme solution, 2.1. Mu.L of ddH ₂ O, 0.5. Mu.L of RPA-F (concentration: 10. Mu. Mol/L), 0.5. Mu.L of RPA-R (concentration: 10. Mu. Mol/L) and 0.5. Mu.L of magnesium acetate solution (concentration: 280 mmol/L).

Wherein the sequence of RPA-F is

5'-ACATCATATCTCCAAAGCACCTTCAACAGACGG-3' (SEQ ID NO: 1), RPA-R has the sequence of

5’-TCTGATTAGGCATGAAAAAGGCTTGTGCTCCC-3’(SEQ ID NO:2)。

The CRISPR/Cas reagent includes ddH2O, NEB buffer 2.1, crRNA, cas12a, and cut probe; preferably, it comprises 4-12. Mu.L NEB buffer 2.1, 2-10. Mu.L crRNA, 2-10. Mu.L Cas12a and 0.2-1.2. Mu.L cut probe (concentration 10. Mu. Mol/L); more preferably, it comprises 51.2. Mu.L ddH2O, 8. Mu.L NEB bufferr2.1, 5. Mu.L crRNA (1. Mu. Mol/L concentration), 5. Mu.L Cas12a (1. Mu. Mol/L concentration) and 0.8. Mu.L cut probe (10. Mu. Mol/L concentration);

wherein the crRNA has the sequence of

5'-UAAUUUCUACUAAGUGUAGAUUGGAAGGCAGCUCCCAAAAC-3' (SEQ ID NO: 3), the sequence of the cut probe is 5'-AGTACCGATAGATACAGAC-3' (SEQ ID NO: 4).

The running buffer included 4 XSSC, 1% BSA and 0.05% Tween20.

The genome of the sample to be detected is extracted by using a commercial genome extraction kit.

The test paper board detection line (T line) is sprayed with a compound after incubation and combination of streptavidin and a TCP probe, and the sequence of the TCP probe is as follows: the spraying of the 5'-CTATCGGTACTATACA-/Biotin/-3' (SEQ ID NO: 5) on the quality control line (C line) is a complex after incubation and combination of streptavidin and a CCP probe, the CCP probe sequence is: 5'-/Biotin/-ATACAGAC-3' (SEQ ID NO: 6).

Before use, 2.5. Mu.L of AuNPs-DNA, which has the DNA sequence 5'-/SH/-TTTTTTTTGTCTGTAT-3' (SEQ ID NO: 7), was pre-dropped onto the binding pad of LFAs on the test paper plate.

In the third step, a dropper is used to blow the liquid in the capillary into the sample tube. Due to capillary forces, the mixture in the sample tube migrates upward along the LFAs. After 3min, the red bands of T line and C line were visually observed, and the whole detection process was less than 35min.

Compared with the prior art, the invention has at least one of the following beneficial effects:

(1) The meat detection sensitivity and specificity can be improved, the integrated coherent operation of the integrated device can be realized, and the requirements of quick, simple, sensitive and specific on-site detection on whether pork is adulterated in halal meat food or not are met;

(2) High sensitivity: the detection sensitivity is improved by establishing a dual signal amplification mechanism of the cooperation of the RPA and the CRISPR/Cas12 a;

(3) High specificity: the CRISPR/Cas12a is used for realizing the secondary control of the specificity, so that the detection specificity is greatly improved;

(4) Low cost: the cost of the test strip is reduced through antibody-free labeling, and visual presentation of results is realized;

(5) The operation is simple and convenient: the integrated device for realizing the RPA, CRISPR/Cas12a and LFAs integrated coherent operation is developed in a matched manner, so that the whole detection process is completed in the next operation on the basis of not opening a sample tube cover, thereby avoiding the pollution of aerosol, reducing the false positive probability of detection, realizing the temperature controllability by adopting an electric water heating tank, and ensuring that the RPA and the CRISPR/Cas12a are carried out at a constant temperature.

In the invention, the technical schemes can be mutually combined to realize more preferable combination schemes. Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and drawings.

Drawings

The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, like reference numerals being used to refer to like parts throughout the several views.

FIG. 1 is a schematic diagram (I) of a halal meat product detection device (a test strip board is positioned on a slot) according to an embodiment;

fig. 2 is a schematic structural diagram (two) of a halal meat food detection device (the test paper board is located on the slot) in the specific embodiment;

FIG. 3 is a cross-sectional view of a Islamic meat product detection device (test strip plate positioned on slot) in an embodiment;

FIG. 4 is a schematic structural diagram of a halal meat product detection device (a test strip plate is positioned in a sample tube) according to an embodiment;

FIG. 5 is a cross-sectional view of a Islamic meat product detection device (dipsticks in sample tubes) in an embodiment;

FIG. 6 is a partial exploded view of a halal meat product detection device in an embodiment;

fig. 7 is a schematic structural diagram (a) of a fixing post in the embodiment;

fig. 8 is a schematic structural view of a fixing post in the embodiment (ii);

fig. 9 is a schematic structural view of a fixing post in the embodiment (iii);

fig. 10 is a schematic structural view of a fixing post in the embodiment (iv);

FIG. 11 is a schematic view of the structure of the assembly plate in the embodiment;

FIG. 12 is a schematic view showing a partial structure of a heater in the embodiment;

FIG. 13 is a schematic of sensitivity for detecting pork adulteration in halal in an embodiment;

FIG. 14 is a schematic diagram showing the specificity of detecting pork adulteration in Islamic foods in an embodiment.

Reference numerals:

1-a detection assembly; 11-sample tube; 111-annular protrusions; 12-fixing columns; 121-a slot; 122-perforating; 123-column part; 123 a-a card plate portion; 124-floor section; 124 a-a slot portion; 124 b-a jack; 124 c-a slider; 13-capillary; 14-test paper board; 2-a heater; 21-a heating chamber; 211-limiting strips; 212-a first fitting port; 22-a storage chamber; 221-a storage cover; 222-a second fitting port; 23-a heating module; 3-assembling plates; 31-an assembly part; 311-sliding grooves; 32-identification area.

Detailed Description

The following detailed description of preferred embodiments of the invention is made in connection with the accompanying drawings, which form a part hereof, and together with the description of the embodiments of the invention, are used to explain the principles of the invention and are not intended to limit the scope of the invention.

In describing embodiments of the present invention, it should be noted that, unless explicitly stated and limited otherwise, the term "coupled" should be interpreted broadly, for example, as being fixedly coupled, as being detachably coupled, as being integrally coupled, as being mechanically coupled, as being electrically coupled, as being directly coupled, as being indirectly coupled via an intermediate medium. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.

The terms "top," "bottom," "above … …," "below," and "on … …" are used throughout the description to refer to the relative positions of components of the device, such as the relative positions of the top and bottom substrates inside the device. It will be appreciated that the devices are versatile, irrespective of their orientation in space.

The working surface of the invention can be a plane or a curved surface, and can be inclined or horizontal. For convenience of explanation, the embodiments of the present invention are placed on a horizontal plane and used on the horizontal plane, and thus "up and down" and "up and down" are defined.

Example 1

The embodiment discloses a halal meat food detection device, as shown in fig. 1-12, including:

at least one detection assembly 1, wherein the detection assembly 1 comprises a sample tube 11, a fixed column 12, a capillary tube 13 and a test paper board 14, and the fixed column 12 is detachably covered on the sample tube 11 so as to be capable of opening or closing the sample tube 11 in an operable manner; the dipstick 14 is operably located on the fixed column 12 or falls from the fixed column 12 into a sample tube; the capillary tube 13 is wound on the fixed column 12, one end of the capillary tube 13 is positioned outside the sample tube 14, the other end of the capillary tube 13 extends into the sample tube 14 so as to be capable of operatively injecting the liquid in the capillary tube 13 into the sample tube, and the capillary tube 13 is used for containing a reagent reacting with a sample to be detected;

the heater 2 is provided with a heating cavity 21, and at least the bottom of the sample tube 11 can extend into the heating cavity 21 so as to prepare the sample tube 11 for heating in the heating cavity 21.

The halal meat food detection device (hereinafter referred to as detection device) of the present invention integrates the sample tube 11 for storing the sample to be detected, the capillary tube 13 carrying the reaction agent, and the chromogenic test paper board 14 for displaying the reaction result together through the fixing column 12, which is convenient for the user to perform the detection operation more simply and conveniently.

The fixing column 12 is provided with a slot 121 matched with the test paper board 14, the slot 121 penetrates through two ends of the fixing column 12, specifically, the slot 121 penetrates through the upper end and the lower end of the fixing column 12, and when the fixing column 12 is installed on the sample tube 11, the slot 121 is communicated with the sample tube 11. Preferably, the slot 121 vertically penetrates through the upper and lower ends of the fixed column 12, and an extension line of the slot 121 is parallel to a center line of the fixed column 12.

The opposite side walls of the slot 121 are respectively provided with a through hole 122 for the pin to pass through.

When the test paper board 14 is located on the fixing column 12, the pins are located in the two through holes 122, and the bottom of the test paper board 14 is inserted into the slot 121 and abutted against the pins so as to fix the test paper board 14 in the slot 121; after the pins are removed from the through holes 122, the test strip plate 14 falls from the fixing column 12 into the sample tube 11.

Preferably, the pin is a steel pin.

In order to prevent the test paper board 14 from being inadvertently dropped into the sample tube 11 when being positioned on the fixing column 12, the pin can completely bet the slot 121 when being positioned between the two through holes 122, i.e. the upward or downward projection of the pin in the two through holes 122 can cover the upper opening and the lower opening of the slot 121, so as to prevent the test paper board 14 from dropping from the gap between the pin and the inner wall of the slot 121, and ensure that the bottom of the test paper board 14 is always abutted with the pin when the pin is positioned between the two through holes 122, i.e. the test paper board 14 is limited in the slot 121 and does not drop into the sample tube.

Preferably, the length of the test strip 14 is greater than the length of the slot 121 to facilitate handling of the test strip 14 and to avoid the test strip 14 from being trapped in the slot 121 and not being removable.

The fixing column 12 includes a column portion 123 and a bottom plate portion 124, and the column portion 123 is located above the bottom plate portion 124, i.e., a bottom end of the column portion 123 is connected to a top surface of the bottom plate portion 124.

The bottom plate portion 124 is matched with the sample tube 11, the bottom plate portion 124 is detachably covered on the opening of the sample tube 11, specifically, the bottom plate portion 124 is provided with a clamping groove portion 124a, the opening of the sample tube 11 is provided with an annular protrusion 111 matched with the clamping groove portion, the annular protrusion 111 is enclosed outside the opening of the sample tube 11, and at least a part of the annular protrusion 111 is detachably embedded into the clamping groove portion 124a. The clamping groove 124a is provided with a groove with one open side, and the annular protrusion 111 is inserted from the open side of the clamping groove 124a, so that a part of the annular protrusion 111 is embedded into the groove of the clamping groove 124a, and the detachable cover of the bottom plate 124 is arranged on the nozzle of the sample tube 11.

The slot 121 extends from the top end of the column portion 123 to the bottom end of the bottom plate portion 124, the through hole 122 is located at the bottom of the sidewall of the column portion 123, and the connection line of the two through holes 122 perpendicularly intersects with the center line of the slot 121.

In order to fix the capillary tube 13 on the fixed column 12 more stably, the side wall of the column portion 123 is provided with a clamping plate portion 123a, the bottom plate portion 124 is provided with an insertion hole 124b for the capillary tube 13 to pass through, and the insertion hole 124b is communicated with the sample tube 11 when the bottom plate portion 124 is covered on the sample tube 11.

The capillary 13 is engaged with the chuck plate portion 123a, and is wound around the cylindrical portion 123 in a downward rotation, and the tip of the capillary 13 extends into the sample tube 11 from the insertion hole 124 b.

For easy detachment of the capillary tube 13, the shape of the clamping plate portion 123a is "L" shape, that is, the clamping plate portion 123a includes a transverse plate vertically connected to the side wall of the column portion 123 and a vertical plate parallel to the side wall of the column portion 123, preferably, the top end face of the transverse plate is flush with the top end face of the column portion 123, the length of the vertical plate is s, and the length of the column portion 123 is L, L/4 is less than or equal to s is less than or equal to L/2.

In order to further improve the stability of the capillary tube 13 on the fixing post 12, the fixing post 12 is preferably non-cylindrical, in this embodiment, the fixing post 12 is similar to an "R", specifically, two opposite outer side walls of the slot 121 extend outwards to form an extending plate, the two extending plates and the outer wall of the slot 121 jointly limit a hollow semi-open cylindrical space, and the top ends and the bottom ends of the two extending plates are respectively provided with a limiting block to limit the winding space of the capillary tube 13.

The number of the insertion holes 124b on the bottom plate portion 124 is equal to the number of the capillaries 13, and all the insertion holes 124b may be distributed on one side of the column portion 123 or may be distributed on both sides of the column portion 123.

The number of capillaries 13 may be appropriately set on the detection assembly 1 according to the type of the sample to be detected, i.e., the detection assembly 1 may include a plurality of capillaries 13, and each capillary 13 preferably contains a different reagent required for detection.

To facilitate distinguishing between the reagents mounted in the different capillaries 13, at least one of the color, pattern, thickness, number, etc. of the capillaries 13 uniquely corresponds to the liquid in the capillaries to prepare for distinguishing the kind of liquid in the capillaries. For example, the liquid in the different capillaries can be distinguished by the capillaries with different colors alone, the liquid in the different capillaries can be distinguished by the capillaries with different patterns, the liquid in the different capillaries can be distinguished by the capillaries with different thicknesses, the liquid in the different capillaries can be distinguished by the capillaries with different numbers, or the liquid in the different capillaries can be distinguished by the combination of two or more of the colors, patterns, thicknesses, numbers and the like of the capillaries, so long as the liquid in the capillaries can be distinguished one by one.

In order to improve the detection efficiency of the detection device, the detection device comprises a plurality of detection assemblies 1 with the same structure so as to be capable of detecting a plurality of samples simultaneously.

Preferably, the detection device further comprises an assembling plate 3, all detection assemblies 1 are detachably mounted on the assembling plate 3, the assembling plate 3 is detachably arranged on the heating cavity 21, and the heating cavity can be opened or closed in an operable mode, namely, the detection assemblies 1 are positioned in the heating cavity 21 through the assembling plate 3 so that part of the sample tube 11 is positioned in the heating cavity 21, and the heating cavity 21 is used for heating the sample tube 11.

In order to facilitate the disassembly of the assembly plate 3, the top of the heating cavity 21 is opened, the assembly plate 3 is embedded at the top of the heating cavity 21, preferably, limit strips 211 are arranged around the inner wall of the heating cavity 21, on one hand, the surface assembly plate 3 is prevented from falling into the heating cavity 21; on the other hand, the assembling plate 3 is fixed in position.

Further for facilitating the assembly and disassembly of the assembly plate 3, a first assembly opening 212 communicated with the top opening of the heating cavity 21 is arranged on the side wall of the heating cavity 21, so that a user can take the assembly plate 3 off the top of the heating cavity 21.

Preferably, after the assembly plate 3 is mounted on top of the heating chamber 21, the top end surface of the assembly plate 3 is flush with the top end surface of the heater 2.

The assembly plate 3 is provided with assembly parts 31 with the same number as the detection assemblies 1, the detection assemblies 1 are detachably arranged in the assembly parts 31, and the detection assemblies 1 are in one-to-one correspondence with the assembly parts 31. Specifically, the bottom plate portion 124 of the fixing post 12 is mated with the assembling portion 31, and the bottom plate portion 124 is detachably disposed on the assembling portion 31.

Preferably, the assembling portion 31 is provided with a sliding groove 311, and the bottom plate portion 124 is provided with a slider 124c that cooperates with the sliding groove 311.

The sliding groove 311 is a U-shaped sliding groove, and the sliding block 124c is a U-shaped sliding block matched with the U-shaped sliding groove. Inserting the slider 124c into the slide groove 311 to integrally assemble the detection assembly 1 to the assembly part 31; the bottom plate 124 is biased to pull out the slider 124c from the slide groove 311, thereby pulling out the entire detection unit 1 from the assembly 31.

In order to facilitate the distinguishing of all the detecting components 1 on the assembling plate 3, the assembling plate 3 is provided with the identifying areas 32 uniquely corresponding to the assembling portions 31, i.e. the number of the identifying areas 32 is equal to the number of the assembling portions 31, and the identifying areas 32 are in one-to-one correspondence with the assembling portions 31. The identification area 32 is provided with a letter, a number, a pattern or the like uniquely corresponding thereto.

Preferably, the heater 2 is also provided with a storage chamber 22 for storing the detection assembly 1 and the assembly plate 3. Further preferably, the storage chamber 22 includes a detachable storage cover 221 for operatively opening or closing the storage chamber 22, the top of the storage chamber 22 is opened, and the storage cover 221 is provided on the top of the storage chamber 22.

In order to facilitate opening the storage cover 221, a second assembling opening 222 is formed in a side wall of the storage cavity 22, the second assembling opening 222 is communicated with the opening of the storage cavity 22, and the first assembling opening 212 is opposite to the second assembling opening 222.

The top end surface of the storage cover 221 is flush with the top end surface of the assembly plate 3.

Preferably, the heater 2 is a water bath heater, and the heating cavity 21 contains water for heating the sample tube. The heater 2 further comprises a heating module 23 for heating the water in the heating chamber 21.

Further preferably, the heater 2 is provided with a control panel, and the control panel is connected with the heating module 23, so that the temperature or power of the heating module 23 can be controlled, and the control of the water bath temperature by a user is facilitated.

The components in the detection assembly 1 and the assembly plate 3 are manufactured by 3D printing.

Example two

The embodiment discloses a halal meat food detection method, which is implemented by the halal meat food detection device disclosed in the first embodiment, and comprises the following steps:

placing an extracted genome of a sample to be detected into a sample tube;

the fixed column is covered on the sample tube;

placing the sample tube into a heating cavity;

In the first step, 9.5 mu L of RPA reagent, 70 mu L of CRISPR/Cas reagent and 120 mu L of running buffer reagent are respectively contained in three capillaries, and the colors of the three capillaries are different, namely the types of the reagents in the capillaries are distinguished through the colors of the capillaries.

The RPA reagent comprises 5.9. Mu.L of resuspended RPA enzyme solution, 2.1. Mu.L of ddH ₂ O, 0.5. Mu.L of RPA-F (concentration: 10. Mu. Mol/L), 0.5. Mu.L of RPA-R (concentration: 10. Mu. Mol/L) and 0.5. Mu.L of magnesium acetate solution (concentration: 280 mmol/L), wherein RPA-FThe sequence of (a) is 5'-ACATCATATCTCCAAAGCACCTTCAACAGACGG-3' (SEQ ID NO: 1), and the sequence of RPA-R is 5 '-TCTGATTAGGCATGAAAAAGGCTTGTGCTCCC-3' (SEQ ID NO: 2).

The CRISPR/Cas reagent comprises 51.2 mu L of ddH2O, 8 mu L of NEB buffer r2.1, 5 mu L of crRNA (with the concentration of 1 mu mol/L), 5 mu L of Cas12a (with the concentration of 1 mu mol/L) and 0.8 mu L of cut probe (with the concentration of 10 mu mol/L), wherein the sequence of the crRNA is 5'-UAAUUUCUACUAAGUGUAGAUUGGAAGGCAGCUCCCAAAAC-3' (SEQ ID NO: 3), and the sequence of the cut probe is 5'-AGTACCGATAGATACAGAC-3' (SEQ ID NO: 4).

The running buffer included 4 XSSC, 1% BSA, and 0.05% Tween20.

In the second step, a lateral flow chromatography test Strip (LFAs) is laid on the test paper board, and comprises a detection line (T line) and a quality control line (C line), wherein the detection line (T line) is sprayed with a compound formed by incubation and combination of streptavidin and a TCP probe, and the sequence of the TCP probe is as follows: the spraying of the 5'-CTATCGGTACTATACA-/Biotin/-3' (SEQ ID NO: 5) on the quality control line (C line) is a complex after incubation and combination of streptavidin and a CCP probe, the CCP probe sequence is: 5'-/Biotin/-ATACAGAC-3' (SEQ ID NO: 6).

If the halal food contains pork components, the test strip has only one red line, namely the T line does not develop color, and the C line develops red. If the halal food does not contain pork components, the test strip shows two red lines, namely T line and C line, which are both red. According to measurement, the qualitative detection line of the method is 0.01g/100g (FIG. 13), and the specificity is good (FIG. 14).

With negative control group (genomic DNA was changed to ddH ₂ And C), comparing the T line of the test group with the T line of the negative control group, if the T line of the test group is light or disappears, judging that pork components are mixed in the halal food, and if the T line of the test group is the same as the T line of the negative control group, judging that the halal food meets the requirements and does not contain the pork components.

The halal meat food detection device and the detection method thereof have the following advantages:

(1) The 3D printing rapid detection device provided by the invention realizes the integration and the continuous operation of RPA, CRISPR/Cas and LFAs, namely, after the previous operation is finished, the tube cover of the sample tube is not required to be opened, the next reaction can be directly carried out, so that the pollution of aerosol is avoided, the false positive probability is reduced, and the portability of detection is realized.

(2) The device realizes the electrothermal temperature control performance of the water tank, and ensures that the RPA and CRISPR/Cas reactions are carried out at the constant temperature of 37 ℃.

(3) The 3D printing rapid detection device is easy and convenient to operate when being used for field detection, and does not need extra large-scale instruments and equipment or professional technicians, and naked eye visual detection results are not needed.

(4) The portable 3D printing rapid detection device does not need an expensive die, can be produced in small batches, and is low in cost.

(5) The detection method provided by the invention has the advantages of rapidness, high sensitivity, good specificity, low cost and visualized result. The method is characterized in that the whole detection process can be completed within 35min, and compared with the traditional method, the detection speed of the whole process is greatly improved; the dual signal amplification of RPA and CRISPR/Cas is integrated, so that the detection sensitivity is improved; and CRISPR/Cas can perform secondary control for specificity, so that the detection specificity is greatly improved; the LFAs marked by the antibody are utilized to realize the low-cost and visual presentation of meat detection results.

(6) On the basis of the invention, by designing different RPA primers and crRNA, the detection device and the detection method thereof can be used for detecting different meats.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Claims

1. A muslims meat product detection device, comprising:

2. The halal meat food detection device of claim 1, wherein the fixed column is provided with a slot matched with the test paper board, and the slot penetrates through two ends of the fixed column;

3. The halal meat product inspection device of claim 2, wherein the stationary post includes a post portion and a base plate portion, the post portion being located above the base plate portion;

4. A muslims meat product testing device in accordance with claim 3 wherein said post portion has a card plate portion and said base plate portion has an insertion hole through which said capillary tube passes;

5. A muslims meat product testing device in accordance with claim 3 wherein said base portion is provided with a slot portion;

6. The halal meat product inspection device of any one of claims 1 to 5, further comprising an assembly plate detachably disposed on the heating chamber, the assembly plate being operable to open or close the heating chamber;

7. The halal meat product inspection device of claim 6, wherein the bottom plate portion of the stationary post mates with the assembly portion, the bottom plate portion being removably disposed to the assembly portion;

8. The halal meat product inspection device of any one of claims 1 to 5, wherein the inspection assembly is provided with a plurality of capillaries, at least one of the color, pattern, thickness, number of the capillaries uniquely corresponding to the capillaries for distinguishing the liquid type within the capillaries.

9. The halal meat product inspection device of any one of claims 1 to 5, wherein the heater is further provided with a storage chamber for storing the inspection assembly.

10. A method for detecting halal meat products, characterized in that it is implemented by a halal meat product detection device according to any one of claims 1 to 9, comprising the steps of:

placing an extracted genome of a sample to be detected into a sample tube;

the fixed column is covered on the sample tube;

placing the sample tube into a heating cavity;