CN115747312A

CN115747312A - Food-borne pathogenic bacteria LFD-RPA detection method and equipment thereof

Info

Publication number: CN115747312A
Application number: CN202211385850.9A
Authority: CN
Inventors: 方美明; 厉佳丽; 张伟; 缪美玲; 秦永胜; 王雅萍
Original assignee: Hangzhou Kuaige Technology Co ltd
Current assignee: Hangzhou Kuaige Technology Co ltd
Priority date: 2022-11-07
Filing date: 2022-11-07
Publication date: 2023-03-07

Abstract

The invention discloses a food-borne pathogenic bacterium LFD-RPA detection method; aiming at the problems of single detection target, inconsistent multi-target synchronous detection efficiency and the like of food-borne pathogenic bacteria in food, the research takes 5 food-borne pathogenic bacteria such as salmonella enteritidis, vibrio parahaemolyticus, listeria monocytogenes, staphylococcus aureus, escherichia coli O157: H7 and the like as research objects, and establishes multiple LFD-RPA synchronous rapid detection by adopting recombinase polymerase nucleic acid amplification technology (RPA) and combining lateral flow chromatography test strips (LFD) through researches such as reaction system optimization, correlation of sample matrixes and amplification efficiency and the like; the efficiency and the accuracy of detection are improved; can provide a multi-target, high-sensitivity, synchronous and rapid detection technology for food-borne pathogenic bacteria and realize multi-bacteria on-site joint detection.

Description

Food-borne pathogenic bacteria LFD-RPA detection method and equipment thereof

Technical Field

The invention belongs to the technical field of food detection, and particularly relates to a food-borne pathogenic bacterium LFD-RPA detection method.

Background

Food-borne pathogenic bacteria are one of the main factors causing food-borne diseases and are concerned in the field of food safety in the world; but the food-borne pathogenic bacteria in the existing food have single detection target and inconsistent multi-target synchronous detection efficiency;

disclosure of Invention

The invention provides a food-borne pathogenic bacterium LFD-RPA detection method for overcoming the defects of the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme: a food-borne pathogenic bacterium LFD-RPA detection method comprises the following steps: s1, preparing a sample required by a test;

s2, selecting a thermostable nuclease gene of staphylococcus aureus, a virulence expression regulatory gene toxR of vibrio parahaemolyticus, a fiber expression regulatory gene fimY of salmonella type 1, a listeria monocytogenes hemolysin gene hlyA and an escherichia coli O157H 7 lipopolysaccharide encoding gene rfbE as a target gene for detection;

s3, preparing a lateral flow chromatography test strip;

s4, performing single RPA amplification of staphylococcus aureus, vibrio parahaemolyticus and salmonella by using an RPA reaction system;

s5, optimizing a multiple RPA reaction system by taking standard plasmids of staphylococcus aureus, vibrio parahaemolyticus and salmonella as DNA templates and performing optimization experiments on primer proportion, reaction temperature, reaction time and Mg < 2+ > concentration;

s6, carrying out a triple LFD-RPA sensitivity experiment and a triple LFD-RPA specificity experiment;

s7, continuously carrying out synchronous rapid detection on 5 food-borne pathogenic bacteria on the basis of experimental research of the triple LFD-RPA;

the preparation of the lateral flow chromatography test strip in the S3 step comprises the following steps: a. the lateral flow chromatography test strip consists of a sample pad, a combination pad, a nitrocellulose membrane (NC membrane), an absorption pad and a bottom plate;

b. placing the bonding pad on a conveying device, firstly placing the bonding pad on a conveying table 1, and fixing the bonding pad on a moving table through an air suction device on the conveying table 1; then driving the mobile station to enter the film dotting instrument; uniformly spraying a colloidal gold solution of the marked digoxin monoclonal antibody of mu g/mL on the combination pad by using a membrane spotting instrument; then the mobile station is driven to enter the transition table again, and the bonding pad is transmitted to the workbench on the transition table through the butt joint device; starting a driving device to drive the rotating disc to rotate 90 degrees, so that the workbench is conveyed into the dehumidifying device to be dehumidified; the dehumidification treatment comprises a top plate arranged in the transition table, a ventilation pipeline arranged in the top plate, an air inlet arranged on the ventilation pipeline, a dehumidification mechanism arranged in the air inlet pipeline, an air outlet arranged on the top plate, a rotating roller arranged in the transition table and a fan arranged at the upper end of the rotating roller; driving a rotating roller to rotate, and starting a fan to suck air from an air inlet; air in the working cavity is sucked into the dehumidifying mechanism for dehumidifying; then enters the working cavity again from the air outlet to form circulation; then the driving device drives the rotating disc to rotate for 90 degrees again; heating the working cavity to 37 ℃; the driving device is started again to drive the rotating disc 13 to rotate for 90 degrees to be in butt joint with the drying box, the bonding pad is conveyed into the drying box, and the temperature is 37 DEG C

Drying for 12h;

c. coating one strip on NC film a test line and an automatic control line; the test line (T) is coated with 0.65mg/mL biotin monoclonal antibody, and the automatic control line (C) is coated with 1mg/mL goat anti-mouse polyclonal antibody;

d. placing the marked bonding pad and the NC membrane in an oven, and drying at 37 ℃ for 12h;

the transition table is arranged to carry out pretreatment before heating, so that the drying box is not influenced by the external environment when being opened every time, and the drying effect of the bonding pad is improved; the detection effect is facilitated; the fixation of the bonding pad is realized through the arrangement of the air suction device, so that the bonding pad is more stable when the colloidal gold solution is sprayed; the arrangement of the butt joint device can ensure that the bonding pad sprayed with the colloidal gold solution can stably enter a transition table, thereby further carrying out pretreatment before drying; the drying stability is improved; the driving device can drive the rotating disc to intermittently rotate for 90 degrees, so that the stability of conveying the combined pad can be improved; the heater can preheat the bonding pad before the bonding pad enters the drying box, so that the bonding pad can adapt to the environment in the drying box on one hand, and the environment in the working cavity can be the same as that of the drying box on the other hand; when the combination pad enters the drying box, the environment in the drying box is changed, so that uneven drying is avoided; the arrangement of the rotating disc can drive the bonding pad to intermittently rotate by 90 degrees, so that the treatment effect on the bonding pad is improved; the air duct is arranged to suck air into the strong working cavity which is positioned under the air duct, and the air in the working cavity is dehumidified; the working cavities at two adjacent sides cannot be influenced; the stability of the treatment of the bonding pad is improved; the dehumidification mechanism can dehumidify the air in the working cavity, so that the environment in the drying box is not influenced when the dehumidifying mechanism is in contact with the drying box, and the stability is improved; different work is made to a plurality of combination pads through the setting of working chamber, has improved the efficiency that the combination pad was handled.

The dehumidifying device also comprises a shutter group arranged below the ventilating duct, butt joints arranged on the top plate and the rotating disc, a piston block arranged in the butt joints, a wind shield arranged on the piston block, a plurality of turnover plates arranged on the wind shield, a moving rod arranged on the shutter group and a wind shield spring arranged on the wind shield; when the fan starts, the air inlet starts to extract air from the working cavity; entering into a ventilation pipeline and then carrying out dehumidification treatment; further processed wind blows to the wind shield from the air outlet, and then the wind passing through the wind shield blows the turnover plate to rotate freely; further changing the wind pressure on the wind shield to drive the wind shield to stretch; at the moment, the piston block moves in the butt joint port, drives the moving rod to move in a reciprocating mode, and simultaneously drives the louver group to swing.

When air enters the air inlet, the air can be divided into three parts through the louver group, so that uniform air suction in the working cavity is realized; the air exchange effect is improved; when wind blows into the working cavity from the air outlet, the wind shield can block and guide the wind, so that the wind is prevented from directly blowing to the bonding pad, and the stability of the bonding pad is improved; the arrangement of the turnover plate realizes that the wind blowing to the wind shield can automatically rotate, so that the wind pressure of the wind on the wind shield is changed; the short-distance movement of the wind shield driven by wind is further realized; along with the movement of the wind shield, the piston block can be driven to move in the butt joint port, so that air pressure is generated in the butt joint port; further driving the movable rod to transversely reciprocate, and then driving the louver board group to turn over; the air energy can uniformly enter the air inlet when air suction is carried out, and the uniformity of air exchange in the working cavity is improved; further improving the stability of drying.

The dehumidifying mechanism comprises a guide groove arranged in the ventilating duct, a plurality of push blocks arranged on the guide groove, two connecting rods arranged on each push block, a steam-water separating plate arranged on the connecting rods, a moving plate arranged on the steam-water separating plate, a filtering hole arranged on the moving plate, absorbent cotton arranged on the moving plate, a water discharging hole arranged in the top plate, an air guide plate arranged on the air inlet, a lifting block arranged in the rotating disk, a first convex block arranged on the lifting block, a second convex block arranged at the bottom of the fan, a lifting spring arranged on the lifting block and a pull rope arranged on the lifting block; when entering from the air inlet, the wind passes through the wind deflector; then the wind is dispersed between the adjacent steam-water separation plates under the action of the wind deflectors; separating water and gas by a steam-water separation plate; further along with the rotation of the fan, the first lug abuts against the second lug to drive the lifting block to move up and down; thereby pulling the push block to move, further connecting rods begin to fold, the absorbent cotton is driven to fold, and water in the absorbent cotton is squeezed into the diversion trench; then water is pushed into the drainage hole along with the movement of the push block.

The water can be guided by the guide groove, so that the water is prevented from flowing back into the working cavity; the water can be pushed through the arrangement of the push block; thereby improving the stability of drainage; the connecting rods can drive the push blocks to synchronously move; air and moisture in the working cavity can be separated through the arrangement of the steam-water separation plate, so that the drying effect is improved; when the push block moves leftwards, the moving plate is connected with the steam-water separation plate; at the moment, water in the water absorption cotton can flow into the diversion trench along the moving plate; the dewatering effect is improved; when the push block moves rightwards, the movable plate is separated from the steam-water separation plate through the absorbent cotton, so that air can be in full contact with the steam-water separation plate, and the steam-water separation uniformity is improved; the water on the steam-water separation plate can be timely treated through the arrangement of the water absorption cotton, so that the water absorption stability is further improved; the air deflector is arranged to enable the air to be fully contacted with the water-vapor separation plate, so that the stability of steam-water separation is improved; when the rotating roller starts to rotate, the first lug and the second lug intermittently abut against each other; the lifting plate is driven to move up and down, so that the pushing block can be pulled to move intermittently; the water is automatically discharged out of the equipment during steam-water separation, and the drying stability is improved.

The driving device comprises a driving cavity arranged at the bottom of the transition table, a driving disc arranged on the rotating roller, a driving rod arranged on the driving disc, a first boss arranged on the driving disc, a second boss arranged at the bottom of the rotating disc, a driving spring connected with the first boss and the second boss, a chute arranged at the side edge of the rotating disc, four clamping blocks arranged on the side wall of the driving cavity, a clamping block spring arranged on the clamping block, a driving gear arranged at the bottom of the driving disc, an eccentric block arranged on the rotating roller, a sleeve ring arranged on the eccentric block, a first chute arranged at the right side of the driving cavity, a first sliding block arranged on the first chute, a hinge rod arranged on the first sliding block, a moving chute arranged at the left side of the driving cavity, a second chute arranged on the moving chute, a second sliding block arranged on the second chute and a connecting rod arranged on the second sliding block; when the rotating roller rotates, the eccentric block is driven to start rotating, and then the lantern ring is driven to start rotating; firstly, the first sliding block is driven to move through the hinged rod and clamped between two teeth of the driving gear; the second sliding block is also embedded between two teeth of the driving gear along with the rotation, and the first sliding block is separated from the driving gear; the second sliding groove is driven to move in the moving groove under the action of the hinge rod; at the moment, the second sliding block drives the driving gear to rotate by a distance of one tooth number; the driving disc is further driven to rotate intermittently, and when the driving disc rotates 90 degrees, the driving rod is pressed against one clamping block; the clamping block is separated from the chute, and then the rotating disc is pulled to rotate under the action of the driving spring; so that the chute is embedded with the next clamping block to limit the rotating disc.

Through the arrangement of the driving spring, when the clamping block is separated from the chute, the rotating disc can be quickly pulled to rotate, so that the clamping block is buckled with the chute again; thereby ensuring that the rotating disc can rotate 90 degrees quickly; the stability of the bonding pad treatment is improved; the driving rod can intermittently rotate along with the driving disc, so that the time for the bonding pad to enter the working cavity is ensured, and the stability is improved; on the other hand, the clamping block can be pressed to stretch out and draw back, so that the clamping block is separated from the chute; the stability of the rotation of the rotating disc is improved; the arrangement of the clamping block and the chute can ensure that the rotating disc rotates by 90 degrees all the time, so that the stability of the treatment of the combined pad is improved; the lantern ring can be driven to move along with the first sliding groove through the arrangement of the eccentric block; the driving gear can be limited through the arrangement of the first sliding block, so that the driving gear rotates towards one direction all the time; the lantern ring can swing along with the rotation of the eccentric block through the arrangement of the hinge rod; along with the swinging of the lantern ring, the second sliding block can drive the driving gear to rotate by the distance of one tooth; thereby achieving intermittent rotation of the drive gear.

The air suction device comprises a conveying belt arranged on the conveying table, a plurality of air vents arranged on the conveying belt, an air suction hole arranged in the conveying table, an air extractor arranged in the air suction hole, an air channel arranged in the side wall of the conveying table, a butt joint pipe arranged on the air channel, an inclined hole arranged on the air channel and an electromagnet arranged on the side wall of the conveying table; starting an air extractor to suck air into the air suction holes, wherein the air suction holes enable the conveying belt to be at negative pressure due to the action of the air suction holes; when the bonding pad is placed on the conveyor belt, the bonding pad is sucked by the vent hole; when the conveying table is in butt joint with the transition table, the butt joint device is started to blow air into the vent groove; simultaneously driving the conveyor belt to start to convey the combined pad; at the moment, the gas is blown out from the inclined hole, so that the head end of the bonding pad is blown; and then transferred into the working chamber.

The combination pad is driven to move through the arrangement of the conveyor belt, so that the treatment efficiency of the combination pad is improved; the device can fix the bonding pad on the conveying belt, thereby improving the processing stability of the bonding pad; the head end of the bonding pad can be tilted upwards when the bonding pad is conveyed through the inclined holes, and other parts are fixed on the conveying belt; the stability of the bond pad entering the working chamber is improved.

The butt joint device comprises a second conveyor belt arranged on the workbench, a magnet arranged on the second conveyor belt, a gas storage cavity arranged at the bottom of the transition table, a through hole arranged on the rotating disc, a butt joint hole arranged on the side edge of the gas storage cavity, a baffle arranged at the right end of the butt joint hole, a push rod arranged on the baffle, a push rod spring arranged on the push rod, a piston plate arranged in the gas storage cavity, a piston spring arranged on the piston plate, an inflating pipe arranged at the bottom of the gas storage cavity, an inflating block arranged on the inflating pipe, an inflating spring arranged on the inflating block and a one-way valve arranged in the inflating pipe; when the second sliding block is clamped into the driving gear, the second sliding block can be pressed against the inflating block to move; then, starting to pump air into the air storage cavity; when the butt joint pipe is in butt joint with the butt joint hole, the push rod is driven to move; then driving the baffle to move; at the moment, the gas in the gas storage cavity enters the butt joint pipe from the butt joint hole; simultaneously, the piston plate starts to suck air into the through hole, so that the second conveying belt is in negative pressure; in addition, when the conveying table is in butt joint with the transition table, the electromagnet is started to attract the magnet; driving the second conveyor belt to convey; and feeding the bonding pad.

The electromagnet can be arranged in the west direction through the arrangement of the magnet, so that the second conveyor belt can be driven to rotate; the stability of the bond pad transfer is improved; the butt joint with the butt joint pipe is realized through the arrangement of the butt joint holes, so that the gas in the gas storage cavity can enter the vent groove; the air pumping in the air storage cavity is realized through the arrangement of the air pumping block; on the other hand can with the second slider counterbalance reset back to drive gear spacing, prevent that drive gear from reseing.

In conclusion, the invention has the following advantages: the invention takes 5 food-borne pathogenic bacteria such as salmonella enteritidis, vibrio parahaemolyticus, listeria monocytogenes, staphylococcus aureus, escherichia coli O157, H7 (Escherichia coli O157, H7) and the like as research objects, adopts recombinase polymerase nucleic acid amplification technology (RPA) and combines lateral flow chromatography test strips (LFD), and establishes multiple LFD-RPA synchronous rapid detection through researches such as reaction system optimization, correlation of sample matrix and amplification efficiency and the like; the efficiency of manufacturing the lateral flow chromatography test strip can be improved when the lateral flow chromatography test strip is prepared; the drying effect of the bonding pad is improved.

Drawings

FIG. 1 is a schematic structural diagram of the present invention.

Fig. 2 is a front view of the present invention.

Fig. 3 is a left side view of the present invention.

Fig. 4 is a cross-sectional perspective view of fig. 3 taken along B-B of the present invention.

Fig. 5 isbase:Sub>A cross-sectional perspective view alongbase:Sub>A-base:Sub>A of fig. 2 of the present invention.

Fig. 6 is a cross-sectional view taken along C-C of fig. 3 of the present invention.

FIG. 7 is a partial view taken at A of FIG. 4 in accordance with the present invention.

Fig. 8 is a partial view of the invention at B in fig. 4.

Fig. 9 is a partial view of the invention at C of fig. 5.

Fig. 10 is a partial view of the invention at D in fig. 5.

Fig. 11 is an exploded view of the driving device of the present invention.

Fig. 12 is an exploded schematic view of the dehumidification mechanism of the present invention.

Detailed Description

As shown in fig. 1-12, a food-borne pathogenic bacteria LFD-RPA detection method comprises the following steps: s1, preparing a sample required by a test;

s3, preparing a lateral flow chromatography test strip;

s4, performing single RPA amplification on staphylococcus aureus, vibrio parahaemolyticus and salmonella by using an RPA reaction system;

s5, optimizing a multiple RPA reaction system by taking standard plasmids of staphylococcus aureus, vibrio parahaemolyticus and salmonella as DNA templates through an optimization experiment of primer proportion, reaction temperature, reaction time and Mg2+ concentration;

s6, performing a triple LFD-RPA sensitivity experiment and a triple LFD-RPA specificity experiment;

wherein, the preparation of the lateral flow chromatography test strip in the step S3 comprises the following steps:

a. the lateral flow chromatography test strip consists of a sample pad, a combination pad, a nitrocellulose membrane (NC membrane), an absorption pad and a bottom plate;

b. placing the bonding pad on a conveying table 1, and fixing the bonding pad on a moving table 1 through an air suction device 2 on the conveying table 1; then the mobile station 1 is driven to enter the film dotting instrument; spraying 10 mu g/mL of colloidal gold solution of labeled digoxin monoclonal antibody on the binding pad uniformly by using a membrane spotting instrument 10; then the mobile station 1 is driven to enter the transition station 11 again, and the bonding pad is transmitted to the workbench 12 on the transition station 11 through the butt joint device 3; starting the driving device 4 to drive the rotating disc 13 to rotate 90 degrees, so that the workbench 12 is conveyed into the dehumidifying device 5 for dehumidifying; the dehumidification treatment 5 comprises a top plate 51, a ventilation pipeline 52, an air inlet 53, a dehumidification mechanism 6, an air outlet 54, a rotating roller 55 and a fan 56; the top plate 51 is fixedly arranged on the transition table 11; the ventilation duct 52 is arranged on the top plate 51; the air inlet 53 is formed in the rightmost end of the ventilation pipeline 52; the dehumidification mechanism 6 is arranged in the ventilation pipeline 52; the air outlet 54 is arranged on the top plate 51; the rotating roller 55 can be rotatably embedded at the bottom of the transition table 11 and is driven by a motor; the fan 56 is provided on the rotating roller 55; the rotation roller 55 is driven to rotate, and then the fan 56 is started to start sucking air from the air inlet 53; air in the working chamber 14 is sucked into the dehumidifying mechanism 6 for dehumidifying; then enters the working chamber 14 again from the air outlet 54 to form a circulation; then the driving device 4 drives the rotating disc 13 to rotate by 90 degrees again; heating the working chamber 14 to 37 ℃; the driving device 4 is started again to drive the rotating disc 13 to rotate for 90 degrees to be in butt joint with the drying box 16, the bonding pad is sent into the drying box 16, and drying is carried out for 12 hours at 37 ℃;

c. coating a test line and an automatic control line on the NC film; the test line (T) is coated with 0.65mg/mL biotin monoclonal antibody, and the automatic control line (C) is coated with 1mg/mL goat anti-mouse polyclonal antibody;

in S2, the vibrio parahaemolyticus RPA upstream primer is labeled Biotin, the escherichia coli O157: H7 upstream primer is labeled FAM, the Listeria monocytogenes upstream primer is labeled TAMRA, the salmonella RPA upstream primer is labeled Digoxin, the staphylococcus aureus RPA upstream primer is labeled Hex, and five groups of RPA downstream primers are labeled Cy5.

S5, preparing a 10-time concentration gradient series bacterial liquid of staphylococcus aureus, vibrio parahaemolyticus and salmonella, obtaining the concentration of the bacterial liquid by a plate counting method, extracting genome DNA, and using the genomic DNA for a triple LFD-RPA sensitivity analysis experiment; the specificity of the multiplex LFD-RPA method is analyzed by detecting staphylococcus aureus, vibrio parahaemolyticus, salmonella, legionella pneumophila, vibrio cholerae, escherichia coli O157H 7, shigella, listeria monocytogenes, enterobacter sakazakii and the like.

In the S5, five different groups such as Hex, digoxin, TAMRA, FAM, biotin and the like are respectively marked at the upstream through different pathogenic bacterium specific primers, and Cy5 groups are marked at the downstream, so that the simultaneous detection of 5 target food-borne pathogenic bacteria is realized.

As shown in fig. 1-12, the conveying apparatus further comprises a base 100, a conveying table 1, an air suction device 2, a transition table 11, a docking device 3, a worktable 12, a driving device 4, a rotating disc 13, a heater 15 and a drying box 16; the conveying table 1 is movably embedded on the base 100, and the conveying table 1 is driven by a motor; the air suction device 2 is arranged on the conveying table 1; the cross section of the transition table 11 is circular and is arranged on the left side of the base 100; the workbench 12 is arranged on the rotating disc 13 and is positioned in the working cavity 14; the driving device 4 is arranged in the transition table 11; the rotating disc 13 is rotatably embedded on the transition table 11; the number of the working chambers 14 is 4, and the working chambers are uniformly arranged on the rotating disk 13 along the circumferential direction of the rotating disk 13; the film dotting instrument 10 is arranged on the base 100 and is positioned in front of the transition table 11; the heater 15 is arranged in the transition table 11; the drying box 16 is arranged on one side of the transition table 11, and an electromagnet is arranged at the connecting part of the drying box 16 and the transition table 11.

As shown in fig. 7, the dehumidifying apparatus 5 further includes a louver group 57, a pair of interfaces 58, a piston block 59, a wind deflector 50, a turning plate 501, a moving rod 502, and a wind deflector spring 508; the louver group 57 is arranged at the bottom of the top plate 51 in a swinging manner and is positioned below the ventilation pipeline 52; the butt-joint ports 58 are respectively arranged on the top plate 51 and the rotating disc 13; the piston block 59 is movably arranged in the butt joint port 58; the wind deflector 50 is movably embedded on the rotating disk 13 and is positioned in front of the air outlet 54; the wind shield 50 is fixedly connected with the piston block 59; the overturning plates 501 are provided with a plurality of overturning plates which are embedded on the wind shield 50 in an overturning way; the movable rod 502 is arranged on the louver group 57, and one end of the movable rod is movably embedded on the inner wall of the top plate 51; the other end is movably embedded in the butt joint port 58; the wind deflector spring 508 connects the wind deflector 50 and the rotary disc 13.

As shown in fig. 7-8 and 12, the dehumidifying mechanism 6 includes a guiding gutter 61, a pushing block 62, a connecting rod 63, a steam-water separating plate 64, a moving plate 65, a filtering hole 66, absorbent cotton 67, a water discharging hole 68, an air guiding plate 69, a lifting block 503, a first bump 504, a second bump 505, a lifting spring 506, and a pulling rope 507; the diversion trench 61 is arranged at the bottom of the ventilation pipeline 52; the push block 62 is slidably embedded in the diversion trench 61; the number of the connecting rods 63 is 2, and one ends of the 2 connecting rods 63 are hinged together; and is arranged on each push block 62; the steam-water separation plate 64 is hinged on the connecting rod 63; the steam-water separation plate 64 is directly purchased from the market; the moving plate 65 is movably embedded on the steam-water separation plate 64; the filtering holes 66 are formed in the moving plate 65; the longitudinal section of the water absorption cotton 67 is V-shaped and is connected with the two adjacent moving plates 65; the drain hole 68 is arranged in the top plate 51, and the drain hole 68 is communicated with the outside; the cross section of the air deflector 69 is in a circular arc shape and is positioned at the upper end of the air inlet; the lifting block 503 can be embedded in the rotating disc 13 and can move up and down; the longitudinal section of the first bump 504 is arc-shaped and is arranged on the lifting block 503; the second bump 505 is arc-shaped in longitudinal section, is arranged at the bottom of the fan 56, and abuts against the first bump 504; the lifting spring 506 is connected with the lifting block 503 and the bottom of the rotating disk 13; the pulling rope 507 connects the lifting block 503 and the pushing block 62.

As shown in fig. 10-11, the driving device 4 includes a driving cavity 40, a driving disk 41, a driving rod 42, a first boss 43, a second boss 44, a driving spring 45, a chute 46, a latch 47, a latch spring 48, a driving gear 49, an eccentric block 401, a collar 402, a first chute 403, a first slider 404, a hinge rod 405, a moving slot 406, a second chute 407, a second slider 408, and a connecting rod 409; the driving cavity 40 is arranged at the bottom of the transition table 11; the driving disc 41 is arranged on the rotating roller 55 and can be rotatably embedded on the inner wall of the driving cavity 40; one end of the driving rod 42 is fixedly arranged on the driving disc 41, and the other end of the driving rod abuts against the clamping block 47; the cross section of one end of the driving rod 42, which is opposite to the clamping block 47, is triangular; the first bump 43 is arranged on the driving disc 41; the second boss 44 is arranged at the bottom of the rotating disc 13; the driving spring 45 is connected with the first boss 43 and the second boss 44; the cross section of the chute 46 is triangular, is arranged on the side of the rotating disc 13 and is buckled with the clamping block 47; the fixture block 47 is provided with 4 blocks, and the cross section of each block is trapezoidal; movably embedded in the side wall of the driving cavity 40; and are uniformly arranged along the circumferential direction of the driving chamber 40; the fixture block spring 48 is connected with the fixture block 47 and the inner wall of the driving cavity 40; the driving gear 49 is arranged at the bottom of the driving disc 41; the eccentric block 401 is fixedly arranged on the rotating roller 55, and the axis of the eccentric block 401 is eccentrically arranged with the axis of the rotating roller 55; the collar 402 is slidably embedded on the eccentric block 401; the first sliding chute 403 is fixedly arranged at the right side of the driving cavity 40 and is positioned below the driving gear 49; the first sliding block 404 is slidably embedded in the first sliding groove 403; one end of the hinge rod 405 is hinged with the first sliding block 404, and the other end is fixedly arranged on the lantern ring 402; the moving groove 406 is arranged on the inner wall of the driving cavity 40; the second sliding chute 407 is movably embedded in the moving chute 406; the second sliding block 408 is movably embedded in the second sliding chute 407; the two ends of the connecting rod 409 are fixedly connected with the second sliding block 408 and the lantern ring 402 respectively.

As shown in fig. 5, the air suction device 2 includes a conveyor belt 21, an air vent 22, an air suction hole 23, an air extractor 24, an air vent 25, a butt joint pipe 26, an inclined hole 27, and an electromagnet 28; the conveyor belt 21 is arranged on the conveying table 1, and the conveyor belt 21 is in a hollow state; a plurality of vent holes 22 are arranged on the conveyor belt 21; the air suction hole 23 is formed in the conveying table 1; the air pump 24 is arranged in the air suction hole 23 and is directly purchased from the market; the vent groove 25 is arranged on the side wall of the conveying table 11; the butt joint pipe 26 is arranged on the vent groove 25; the inclined hole 27 is formed in the vent groove 25; the electromagnet 28 is arranged at the front end of the conveying table 1.

As shown in fig. 9-10, the docking device 3 includes a second conveyor 32, a magnet 33, an air storage chamber 34, a through hole 35, a docking hole 36, a baffle 37, a push rod 38, a push rod spring 39, a piston plate 30, a piston spring 301, an air inflation pipe 302, an air inflation block 303, an air inflation spring 304, and a one-way valve 305; the second conveyor belt 32 is arranged on the workbench 12, and the second conveyor belt 32 has the same structure as the conveyor belt 21; the magnet 33 is arranged at the bottom of the second conveyor belt 32 close to the right end; the gas storage cavity 34 is arranged on the transition table 31; the through hole 35 is formed in the rotating disc 13 and is connected with the air storage cavity 34 and the bottom of the workbench 12; the butt joint hole 36 is arranged on the transition table 11 and is connected with the butt joint pipe and the gas storage cavity 34; the baffle 37 is movably embedded at the right end of the butt joint hole 36; the push rod 38 is fixedly arranged on the baffle 37 and is abutted against the butt joint pipe 26; the push rod spring 39 is connected with the push rod 38 and the butt joint hole 36; the piston plate 30 can be embedded in the air storage cavity 34 in an up-and-down moving mode; the piston spring 301 is connected with the piston plate 30 and the bottom of the air storage cavity 34; the inflating pipe 302 is arranged at the bottom of the air storage cavity 34; the inflating block 303 is movably embedded in the inflating pipe 302 and is abutted against the second sliding block 180; and the pumping block 303 can be embedded between two teeth of the driving gear 49; the inflating spring 304 is connected with the inflating block 303 and the inflating pipe 302; the check valve 305 is disposed in the inflation tube 302.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. A food-borne pathogenic bacterium LFD-RPA detection method is characterized in that: the method comprises the following steps: s1, preparing a sample required by a test;

s3, preparing a lateral flow chromatography test strip;

b. placing the combined pad on a conveying device, sending the combined pad into a drying box (16), and drying for 12h at 37 ℃;

d. the labeled conjugate pad and NC membrane were placed in an oven and dried at 37 ℃ for 12h.

2. The method for detecting food-borne pathogenic bacteria LFD-RPA according to claim 1, which is characterized in that: in S2, the vibrio parahaemolyticus RPA upstream primer is labeled Biotin, the escherichia coli O157: H7 upstream primer is labeled FAM, the Listeria monocytogenes upstream primer is labeled TAMRA, the salmonella RPA upstream primer is labeled Digoxin, the staphylococcus aureus RPA upstream primer is labeled Hex, and five groups of RPA downstream primers are labeled Cy5.

3. The method for detecting food-borne pathogenic bacteria LFD-RPA according to claim 1, which is characterized in that: s5, preparing a 10-time concentration gradient series bacterial liquid of staphylococcus aureus, vibrio parahaemolyticus and salmonella, obtaining the concentration of the bacterial liquid by a plate counting method, extracting genome DNA, and using the genomic DNA for a triple LFD-RPA sensitivity analysis experiment; the specificity of the multiplex LFD-RPA method is analyzed by detecting staphylococcus aureus, vibrio parahaemolyticus, salmonella, legionella pneumophila, vibrio cholerae, escherichia coli O157H 7, shigella, listeria monocytogenes, enterobacter sakazakii and the like.

4. The method for detecting food-borne pathogenic bacteria LFD-RPA according to claim 1, which is characterized in that: in the S7, five different groups such as Hex, digoxin, TAMRA, FAM, biotin and the like are respectively marked at the upstream through different pathogenic bacteria specific primers, and Cy5 groups are marked at the downstream, so that the simultaneous detection of 5 target food-borne pathogenic bacteria is realized.

5. A drying apparatus for drying the conjugate pad of claim 1, wherein:

the method comprises the following steps: the combination pad is fixed on the moving table (1) through an air suction device (2) on the conveying table (1); then the mobile station (1) is driven to enter the film dotting instrument; uniformly spraying a 10 mu g/mL colloidal gold solution of the labeled digoxin monoclonal antibody on the combination pad by using a membrane spotting instrument; then the mobile station (1) is driven to enter the transition station (11) again, and the bonding pad is transmitted to the workbench (12) on the transition station (11) through the butt joint device (3); starting a driving device (4) to drive a rotating disc (13) to rotate (90 degrees), so that the workbench (12) is conveyed into a dehumidifying device (5) for dehumidifying; the dehumidification treatment (5) comprises a top plate (51) arranged in the transition table (11), a ventilation pipeline (52) arranged in the top plate (51), an air inlet (53) arranged on the ventilation pipeline (52), a dehumidification mechanism (6) arranged in the air inlet pipeline (52), an air outlet (54) arranged on the top plate (51), a rotating roller (55) arranged in the transition table (11) and a fan (56) arranged at the upper end of the rotating roller (55); driving a rotating roller (55) to rotate, and then starting a fan (56) to suck air from an air inlet (53); air in the working cavity (14) is sucked into the dehumidifying mechanism (6) for dehumidifying; then enters the working cavity (14) again from the air outlet (54) to form circulation; then the driving device (4) drives the rotating disc (13) to rotate (0 degrees) again, the working cavity (14) is heated to 37 degrees, the driving device (4) is started again to drive the rotating disc (13) to rotate 90 degrees and be in butt joint with the drying box (16), the bonding pad is sent into the drying box (16), and drying is carried out for 12 hours at 37 degrees.

6. The method for detecting food-borne pathogenic bacteria LFD-RPA according to claim 1, which is characterized in that: the dehumidifying device (5) further comprises a shutter group (57) arranged below the ventilating duct (52), a butt joint opening (58) arranged on the top plate (51) and the rotating disc (13), a piston block (59) arranged in the butt joint opening (58), a wind shield (50) arranged on the piston block (59), a plurality of turnover plates (501) arranged on the wind shield (50), a moving rod (502) arranged on the shutter group (57), and a wind shield spring (508) arranged on the wind shield (50); when the fan (56) starts, the air inlet (53) starts to extract air from the working cavity (14); entering a ventilation pipeline (52) and then carrying out dehumidification treatment; further processed wind blows to the wind shield (50) from the air outlet (54), and then the wind passing through the wind shield (50) blows the turnover plate (501) to rotate freely; further changing the wind pressure on the wind shield (50) to drive the wind shield (50) to stretch; at this time, the piston block (59) moves in the butt joint port (58), drives the moving rod (502) to reciprocate, and drives the louver group (57) to swing.

7. The method for detecting food-borne pathogenic bacteria LFD-RPA according to claim 1, which is characterized in that: the dehumidifying mechanism (6) comprises a guide groove (61) arranged in the ventilating duct (52), a plurality of push blocks (62) arranged on the guide groove (61), two connecting rods (63) arranged on each push block, a steam-water separating plate (64) arranged on the connecting rods (63), a moving plate (65) arranged on the steam-water separating plate (64), a filtering hole (66) arranged on the moving plate (65), absorbent cotton (67) arranged on the moving plate (65), a water discharging hole (68) arranged in the top plate (51), an air guide plate (69) arranged on the air inlet (53), a lifting block (503) arranged in the rotating disc (13), a first bump (504) arranged on the lifting block (503), a second bump (505) arranged at the bottom of the fan (56), a lifting spring (506) arranged on the lifting block (503), and a pulling rope (507) arranged on the lifting block (503); when wind enters from the air inlet (53), the wind passes through the wind deflector (69); then the wind is dispersed between the adjacent steam-water separation plates (64) under the action of the wind deflector (53); water and gas are separated by a steam-water separation plate (64); further along with the rotation of the fan, the first lug (504) abuts against the second lug (505) to drive the lifting block (503) to move up and down; thereby pulling the push block (62) to move, further folding the connecting rod (63) to drive the water absorption cotton (67) to fold, and extruding water in the water absorption cotton into the diversion trench (61); water is then propelled into the drain holes (68) as the push block (62) moves.

8. The food-borne pathogenic bacteria LFD-RPA detection method according to claim 1, wherein the driving device (4) comprises a driving chamber (40) disposed at the bottom of the transition table (11), a driving disc (41) disposed on the rotating roller (55), a driving rod (42) disposed on the driving disc (41), a first boss (43) disposed on the driving disc (41), a second boss (44) disposed at the bottom of the rotating disc (13), a driving spring (45) connecting the first boss (43) and the second boss (44), a chute (46) disposed at the side of the rotating disc (13), four blocks (47) disposed on the side wall of the driving chamber (40), a block (48) disposed on the block (47), a driving gear (49) disposed at the bottom of the driving disc (41), an eccentric block (401) disposed on the rotating roller (55), a collar (402) disposed on the eccentric block (401), a first chute (403) disposed at the right side of the driving chamber (40), a first slider (403) disposed on the first chute (404), a moving rod (407) disposed on the first chute (406), and a moving slot (407) disposed on the left side of the driving chamber (406), and a moving rod (406) disposed on the second chute (406) A second sliding block (408) arranged on the second sliding chute (407), and a connecting rod (409) arranged on the second sliding block (408); when the rotating roller (55) rotates, the eccentric block (401) is driven to rotate, and then the lantern ring (402) is driven to rotate; firstly, a first sliding block (404) is driven to move through a hinged rod (405) and is clamped between two teeth of a driving gear (49); the second slider (408) is also embedded between two teeth of the driving gear (49) along with the rotation, and the first slider (404) is separated from the driving gear (49); and under the action of the hinge rod (405), the second sliding chute (407) is driven to move in the moving chute (406); at the moment, the second sliding block (408) drives the driving gear (49) to rotate by the distance of one tooth number; the driving disc (41) is further driven to intermittently rotate, and when the driving disc rotates by 90 degrees, the driving rod (42) is pressed against one clamping block (47); the clamping block (47) is separated from the inclined groove (46), and then the rotating disc (13) is pulled to rotate for 90 degrees under the action of the driving spring (45); so that the inclined groove (46) is embedded with the next fixture block (47) to limit the rotating disc (13).

9. The method for detecting food-borne pathogenic bacteria LFD-RPA according to claim 1, which is characterized in that: the air suction device (2) comprises a conveyor belt (21) arranged on the conveying table (1), a plurality of vent holes (22) arranged on the conveyor belt (21), an air suction hole (23) arranged in the conveying table (1), an air extractor (24) arranged in the air suction hole (23), an air channel (25) arranged in the side wall of the conveying table (1), a butt joint pipe (26) arranged on the air channel (25), an inclined hole (27) arranged on the air channel (25), and an electromagnet (28) arranged on the side wall of the conveying table (1); starting an air extractor (24) to suck air into the air suction holes (23), and enabling the conveyor belt (21) to be at negative pressure due to the action of the air vent holes (22); when the bonding pad is placed on the conveyor belt (21), the bonding pad is sucked by the vent hole (22); when the conveying table (1) is in butt joint with the transition table (11), the butt joint device (3) is started to blow air into the air channel (25); simultaneously driving the conveyor belt (21) to start to convey the bonding pad; at the moment, the gas is blown out from the inclined hole (27), so that the head end of the bonding pad is blown; and then transferred into the working chamber (14).

10. The method for detecting food-borne pathogenic bacteria LFD-RPA according to claim 1, which is characterized in that: the butt joint device (3) comprises a second conveyor belt (32) arranged on the workbench (12), a magnet (33) arranged on the second conveyor belt (32), an air storage cavity (34) arranged at the bottom of the transition table (11), a through hole (35) arranged on the rotary disc (13), a butt joint hole (36) arranged on the side edge of the air storage cavity (34), a baffle (37) arranged at the right end of the butt joint hole (36), a push rod (38) arranged on the baffle (37), a push rod spring (39) arranged on the push rod (38), a piston plate (30) arranged in the air storage cavity (34), a piston spring (301) arranged on the piston plate (30), an air inflating pipe (302) arranged at the bottom of the air storage cavity (34), an air inflating block (303) arranged on the air inflating pipe (302), an air inflating spring (304) arranged on the inflating block (303), and a one-way valve (305) arranged in the inflating pipe (302); when the second sliding block (108) is clamped into the driving gear (49), the second sliding block can be pressed against the inflating block (303) to move; then, starting to pump air into the air storage cavity (34); when the butt joint pipe (26) is in butt joint with the butt joint hole (36), the push rod (38) is driven to move; then the baffle (37) is driven to move; at the moment, the gas in the gas storage cavity (34) enters the butt joint pipe (26) from the butt joint hole (36); simultaneously, the piston plate (30) starts to suck air into the through hole (35) so that the interior of the second conveyor belt (32) is under negative pressure; in addition, when the conveying platform (1) is butted with the transition platform (11), the electromagnet (28) is started to attract the magnet (33); at the moment, the second conveyor belt (32) is driven to convey; and feeding the bonding pad.