An antibiotic resistance gene detection device and a detection method

Abstract

The application discloses an antibiotic resistance gene detection device and a detection method. The detection device comprises a rack and a bottom plate. An outer sleeve is arranged on the bottom plate. A sample groove and a sample injection hole are arranged on the side wall of the outer sleeve. A liquid inlet pipe is further communicated with the bottom of the side wall of the outer sleeve. An inner sleeve is sealingly and rotatably connected to the inner wall of the outer sleeve. A first through hole and a second through hole are arranged on the bottom of the inner sleeve. An upper cover is arranged on the top of the inner sleeve. A lifting mechanism is arranged on the top of the rack. A hollow assembly is arranged on the lifting mechanism. The lower end of the hollow assembly is detachably communicated with the inner sleeve. A rotating assembly for rotating the inner sleeve is arranged on the lower part of the hollow assembly. The application can not only successfully realize the injection of the sample liquid and the detection reagent while fully mixing the sample liquid and the detection reagent by controlling the pressure difference between the inner sleeve and the outside world and the self-rotation speed of the inner sleeve, but also can avoid the generation of bubbles and wall hanging phenomenon, and improve the detection efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to a biochemical detection device and a detection method, in particular to an antibiotic resistance gene detection device and a detection method. BACKGROUND

[0002] The existing antibiotic resistance gene detection principle is to use the nucleic acid in the sample liquid as a template, and use a specific fluorescent probe for antibiotic resistance gene for qPCR reaction, and judge whether the sample liquid contains antibiotic resistance gene fragments according to the qPCR reaction result. The probe method detection reagent mainly includes Gene Expression Master Mix (2x), Nuclease-free PCR-grade water, primers and Taqman probes.

[0003] Before the detection reaction, the above detection reagent needs to be mixed and injected into the multi-well plate, and then the sample liquid is injected, mixed and sealed for reaction. In this process, although there are many existing devices that can realize automatic pipetting, these devices are all from the top of the multi-well plate to add or inject liquid. Because the surface tension of the detection reagent and the sample liquid is large, although the injection process is slow and gentle, bubbles will inevitably be generated at the gun head when the liquid is exhausted, and liquid droplets will also be hung on the inner wall of the reaction hole. The bubbles and the hanging wall phenomenon will lead to uneven reaction conditions in each hole, which seriously affects the accuracy of the experimental results.

[0004] In order to solve the above problems, the current common method is to centrifuge the multi-well plate after sealing the film after injecting the liquid, remove the bubbles and shake the wall liquid droplets into the liquid at the bottom of the reaction hole. The centrifugation step is time-consuming and laborious when detecting a large number of samples, which seriously affects the detection efficiency. How to directly avoid the generation of bubbles and hanging wall phenomenon when the liquid is injected has become a technical problem to be solved. SUMMARY

[0005] The purpose of the present application is to provide an antibiotic resistance gene detection device to solve the problem of easy bubble and wall hanging when the existing device is batch injected. Another purpose of the present application is to provide an antibiotic resistance gene detection method to solve the problem of how to detect antibiotic resistance genes.

[0006] Technical solution: The device for detecting antibiotic resistance genes comprises a rack and a bottom plate detachably connected to the bottom of the rack, an outer sleeve is arranged on the bottom plate, a sample groove is arranged on the sidewall of the outer sleeve, a sample injection hole is arranged at the bottom of the sample groove, a liquid inlet pipe is further communicated with the bottom of the sidewall of the outer sleeve, an inner sleeve is sealingly and rotatably connected to the inner wall of the outer sleeve, a first through hole corresponding to the liquid inlet pipe and a second through hole corresponding to the sample injection hole are arranged at the bottom of the inner sleeve, an upper cover is arranged at the top of the inner sleeve, a lifting mechanism is arranged at the top of the rack, a hollow component for creating a negative pressure environment in the inner sleeve is arranged on the lifting mechanism, the lower end of the hollow component is detachably communicated with the inner sleeve, a rotating component for driving the rotation of the inner sleeve is arranged at the lower part of the hollow component, the lower end of the rotating component is detachably connected with the upper cover, and a driving mechanism is arranged on one side of the rotating component.

[0007] Preferably, the hollow component comprises a lifting plate arranged on the lifting mechanism, an inner cavity is arranged in the lifting plate, a negative pressure pipe is arranged on the lifting plate and communicated with the inner cavity, a hollow base is arranged at the bottom of the lifting plate and communicated with the inner cavity, a cannula is communicated with the lower end of the hollow base, and a jack is arranged on the upper cover and used for inserting the lower end of the cannula.

[0008] Preferably, the rotating component comprises a gear rotatably connected to the outer wall of the hollow base, and an insertion strip is hung on the gear, an insertion slot is arranged on the upper cover and used for inserting the lower end of the insertion strip, a rack is slidingly connected to the bottom of the lifting plate and engaged with the gear, and the driving mechanism is arranged at one end of the rack.

[0009] Preferably, the driving mechanism comprises a connecting strip arranged at one end of the rack, a second electric cylinder is arranged on the lifting plate, and the telescopic rod of the second electric cylinder is fixedly connected with the connecting strip.

[0010] Preferably, the lifting mechanism comprises a first electric cylinder arranged at the top of the rack, the lifting plate is arranged at the lower end of the telescopic rod of the first electric cylinder, and a guide rod is slidingly connected with the top of the rack.

[0011] Preferably, a main pipe is communicated with the liquid inlet pipe, a placing groove is arranged at the bottom of the rack, and the bottom plate is arranged in the placing groove.

[0012] Another aspect of the present application discloses a method for detecting antibiotic resistance genes by using the above device, which comprises the following steps:

[0013] (1) In the initial state, ensure that the liquid inlet pipe is completely dislocated and closed with the first through hole, the sample injection hole is completely dislocated and closed with the second through hole, the negative pressure pipe is connected to the negative pressure pump, the liquid inlet pipe is communicated with the liquid storage tank, the detection reagent is filled in the liquid inlet pipe, the sample liquid is injected into the sample groove, and then the bottom plate is placed to ensure that the jack is located directly below the cannula and the insertion slot is located directly below the insertion strip.

[0014] (2) Lower the lifting plate until the cannula is inserted into the insertion hole and the insertion bar is inserted into the insertion slot, stop lowering the lifting plate, turn on the negative pressure pump, and suck the air in the inner sleeve through the cannula to form a constant negative pressure in the inner sleeve;

[0015] (3) Pull the rack to drive the gear and the insertion bar to rotate around the hollow base, and the insertion bar drives the inner sleeve to rotate synchronously. When the liquid inlet pipe is in communication with the first through hole, and / or the sample liquid injection hole is in communication with the second through hole, the sample liquid and / or the detection reagent flow into the inner sleeve from the bottom of the outer sleeve and the inner sleeve to mix, and the inner sleeve is continuously rotated until the liquid inlet pipe is completely misaligned and closed with the first through hole, and the sample liquid injection hole is completely misaligned and closed with the second through hole, and the liquid injection operation is completed;

[0016] (4) The ratio of the pressure difference between the inner sleeve and the outside and the self-rotation speed of the inner sleeve is controlled to ensure that the rated amount of sample liquid and detection reagent is injected and to avoid liquid splashing and air bubble generation;

[0017] (5) After the liquid injection is completed, the lifting plate is lifted to separate the cannula from the insertion hole and the insertion bar from the insertion slot, and the bottom plate and the outer sleeve thereon are removed and placed in a qPCR instrument for reaction. According to the reaction result, it can be known whether the sample liquid contains the corresponding antibiotic resistance gene.

[0018] Preferably, in step (1), the sample liquid is injected into the corresponding sample groove according to the following sample array position distribution principle:

[0019] According to S1×A1, S1×A2, S1×A3……S1×A m-2 , S1×A m-1 , S1×A m ; S2×A1, S2×A2, S2×A3……S2×A m-2 , S2×A m-1 , S2×A m ; S3×A1, S3×A2, S3×A3……S3×A m-2 , S3×A m-1 , S3×A m ;……S n-1 ×A1, S n-1 ×A2, S n-1 ×A3……S n-1 ×A m-2 , S n-1 ×A m-1 , S n-1 ×A m ; S n ×A1, S n ×A2, S n ×A3……S n ×A m-2 , S nX A m-1 , S n X A m order;

[0020] or S1X A1, S2X A1, S3X A1... S n-2 X A1, S n-1 X A1, S n X A1; S1X A2, S2X A2, S3X A2... S n-2 X A2, S n-1 X A2, S n X A2; S1X A3, S2X A3, S3X A3... S n-2 X A3, S n-1 X A3, S n X A3;... S1X A m-1 , S2X A m-1 , S3X A m-1 ... S n-2 X A m-1 , S n-1 X A m-1 , S n X A m-1 ; S1X A m , S2X A m , S3X A m ... S n-2 X A m , S n-1 X A m , S n X A m order;

[0021] Different sample liquids are sequentially injected into corresponding sample grooves from top to bottom in the longitudinal direction and from left to right in the transverse direction, wherein n is the number of samples to be tested, m is the number of antibiotic resistance genes to be tested, and each sample has no less than 2 technical repeats for each antibiotic resistance gene.

[0022] Preferably, one side of the sample liquid spotting column is provided with a control column, and the control column contains positive and negative controls. The positive and negative controls can be alternately arranged or separately arranged continuously.

[0023] Preferably, the sample liquid contains nucleic acid extracts of samples to be tested and corresponding fluorescent probes of antibiotic resistance genes to be tested.

[0024] Advantages: Compared with the prior art, the present application has the following significant advantages:

[0025] The present application injects liquid from the bottom of the reaction container, avoiding the problems of air bubbles and liquid splashing during pipetting and injection from the upper part. In addition, by controlling the pressure difference between the inner sleeve and the outside and the self-rotation speed of the inner sleeve, two key operating parameters, not only can the injection be successfully implemented while fully mixing the sample liquid and the detection reagent, but also can avoid the generation of air bubbles and wall hanging phenomenon, simplify the operation steps, and improve the efficiency of antibiotic resistance gene detection. In addition, the pressure difference can be generated by forming negative pressure in the reaction container, which can facilitate batch injection operation, effectively ensure the rapid and constant pressure in multiple reaction containers, improve the synchronization and precision of injection operation, and quickly adjust the pressure difference in batches according to the needs, and the adjustment operation is simple and fast. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a schematic diagram of the overall structure of the present application;

[0027] Figure 2 It is a top view of the present application;

[0028] Figure 3 It is Figure 2 A-A sectional view in the present application;

[0029] Figure 4 It is Figure 2 B-B sectional view in the present application;

[0030] Figure 5 It is a schematic diagram of the bottom plate and its attached structure;

[0031] Figure 6 It is a schematic diagram of the lifting plate and its related structure;

[0032] Wherein, 1- rack, 10- bottom plate, 11- first electric cylinder, 12- guide rod, 13- installation groove, 2- outer sleeve, 21- sample groove, 211- sample injection hole, 3- inner sleeve, 31- upper cover, 32- insertion hole, 33- insertion slot, 34- first through hole, 35- second through hole, 4- main pipe, 41- liquid inlet pipe, 5- rack, 51- gear, 52- insertion strip, 53- second electric cylinder, 54- connecting strip, 6- lifting plate, 61- hollow base, 62- insertion pipe, 63- negative pressure pipe, 64- inner cavity;

[0033] Figure 7 It is a sample of 1 x 256 ARGs mode spotting array diagram;

[0034] Figure 8 It is a sample of 8 x 32 ARGs mode spotting array diagram;

[0035] Figure 9 It is a sample of 16 x 16 ARGs mode spotting array diagram;

[0036] Figure 10For 32 samples x 8 ARGs mode spotting array chart;

[0037] Figure 11 For 64 samples x 4 ARGs mode spotting array chart. DETAILED DESCRIPTION

[0038] The technical solutions of the present application are further described below in conjunction with the drawings.

[0039] As Figures 1-6 shown, an antibiotic resistance gene detection device comprises a rack 1, the bottom of the rack 1 is provided with a mounting groove 13, the mounting groove 13 is provided with a bottom plate 10, the mounting groove 13 can help the bottom plate 10 to be quickly positioned and aligned. The bottom plate 10 is provided with an outer sleeve 2, the sidewall of the outer sleeve 2 is provided with a sample groove 21, the bottom of the sample groove 21 is communicated with a sample injection hole 211, the sidewall of the outer sleeve 2 is also communicated with a liquid inlet pipe 41, the inner wall of the outer sleeve 2 is sealingly rotatably connected with an inner sleeve 3, the bottom of the inner sleeve 3 is provided with a first through hole 34 which can be correspondingly communicated with the liquid inlet pipe 41 and a second through hole 35 which can be correspondingly communicated with the sample injection hole 211, the top of the inner sleeve 3 is provided with an upper cover 31, the top of the rack 1 is provided with a lifting mechanism, the lifting mechanism is provided with a lifting plate 6, the inside of the lifting plate 6 is provided with an inner cavity 64, the lifting plate 6 is provided with a negative pressure pipe 63 which is communicated with the inner cavity 64, the bottom of the lifting plate 6 is provided with a hollow base 61 which is communicated with the inner cavity 64, the lower end of the hollow base 61 is communicated with a cannula 62, the upper cover 31 is correspondingly provided with a insertion hole 32 for the lower end of the cannula 62 to be inserted, the outer wall of the hollow base 61 is rotatably connected with a gear 51, the gear 51 is hung with an insertion strip 52, the upper cover 31 is correspondingly provided with an insertion slot 33 for the lower end of the insertion strip 52 to be inserted, the bottom of the lifting plate 6 is slidingly connected with a rack 5 which is engaged with the gear 51, one end of the rack 5 is provided with a connecting strip 54, the lifting plate 6 is provided with a second electric cylinder 53, the telescopic rod of the second electric cylinder 53 is fixedly connected with the connecting strip 54.

[0040] In this embodiment, the top of the rack 5 is slidingly connected with the bottom of the lifting plate 6, one side of the rack 5 is engaged with the gear 51. The rack 5 can drive the gear 51 to rotate around the hollow base 61 in the process of sliding in the horizontal direction, and then drive the upper cover 31 and the inner sleeve 3 to rotate through the insertion strip 52 hung below the gear 51.

[0041] In this embodiment, the outer sleeve 2 and the inner sleeve 3 are both columnar, and the inner sleeve 3 can be bottomed or unbottomed. The outer sleeve 2 and the inner sleeve 3 can be sealingly and slidingly connected through an annular sliding groove and an annular sliding strip.

[0042] In the embodiment, the lifting mechanism comprises a first electric cylinder 11 arranged on the top of the frame 1, and the lifting plate 6 is arranged on the lower end of the telescopic rod of the first electric cylinder 11, and the lifting plate 6 is provided with a guide rod 12 which is in sliding connection with the top of the frame 1.

[0043] In the embodiment, in order to control the synchronous liquid injection of the plurality of outer sleeves 2 and inner sleeves 3, a plurality of liquid inlet pipes 41 are connected to the main pipe 4, and the main pipe 4 is used for unified liquid supply.

[0044] In the initial state, the liquid inlet pipe 41 is completely misaligned and closed with the first through hole 34, and the sample liquid injection hole 211 is completely misaligned and closed with the second through hole 35. The negative pressure pipe 63 is connected to the negative pressure pump, the liquid inlet pipe 41 is connected to the liquid storage tank through the main pipe 4, and an exhaust hole can be arranged at the end of the main pipe 4. The detection reagent in the liquid storage tank is pumped into the main pipe 4 and the liquid inlet pipe 41 through the peristaltic pump, and the air in the main pipe 4 and the liquid inlet pipe 41 is discharged. The detection reagent fills the liquid inlet pipe 41 and the main pipe 4, the peristaltic pump is removed, and the detection reagent is under the atmospheric pressure of the outside world. Then, the sample liquid is injected into the sample groove 21 for standby.

[0045] The bottom plate 10 is placed in the placement groove 13, at this time the jack 32 is just below the insertion pipe 62, and the insertion slot 33 is below the insertion strip 52; the first electric cylinder 11 is opened, the lifting plate 6 is driven to descend, until the insertion pipe 62 is inserted into the jack 32, and at the same time the insertion strip 52 is inserted into the insertion slot 33, the lifting plate 6 is stopped, after the insertion pipe 62 is inserted into the jack 32, the inner sleeve 3 is in sealed communication with the hollow base 61 and the inner cavity 64, the negative pressure pump is opened, and the air in the inner sleeve 3 is sucked through the negative pressure pipe 63, the inner cavity 64, the hollow base 61 and the insertion pipe 62, and a constant negative pressure is formed in the inner sleeve 3;

[0046] The second electric cylinder 53 is opened, the connecting strip 54 is pushed to move outward, the connecting strip 54 drives the rack 5 to slide outward, the rack 5 drives the gear 51 and the insertion strip 52 to rotate around the hollow base 61, the insertion strip 52 drives the inner sleeve 3 to rotate synchronously through the rotating cover 31, until the first through hole 34 of the inner sleeve 3 is in communication with the liquid inlet pipe 41, and the second through hole 35 is in communication with the sample liquid injection hole 211. Under the pressure difference between the inner sleeve 3 and the outside world, the detection reagent is injected into the inner sleeve 3 through the liquid inlet pipe 41 and the first through hole 34, and the sample liquid is injected into the inner sleeve 3 through the sample liquid injection hole 211 and the second through hole 35. In this way, the sample liquid and the detection reagent continuously flow into the inner sleeve 3 from the bottom of the outer sleeve 2 and the inner sleeve 3 and mix. In this process, the inner sleeve 3 is continuously rotated until the liquid inlet pipe 41 is completely misaligned and closed with the first through hole 34, and the sample liquid injection hole 211 is completely misaligned and closed with the second through hole 35, and the liquid injection operation is completed;

[0047] The injection of the rated amount of sample liquid and detection reagent is ensured and liquid splashing and bubble generation are avoided by controlling the communication time length of the liquid inlet pipe 41 with the first through hole 34 and the sample injection hole 211 with the second through hole 35 and the pressure difference between the inner sleeve 3 and the outside world; the greater the pressure difference between the inner sleeve 3 and the outside world, the greater the flow of the injected sample liquid and detection reagent, but excessive flow will lead to splashing or spattering of the sample liquid and detection reagent, which will also generate bubbles and wall hanging. When the flow is too small, the slow injection of the sample liquid and detection reagent will easily lead to uneven mixing of the two, which requires additional oscillation mixing and will also increase the generation of bubbles and wall hanging.

[0048] The formula for calculating the injection flow according to the pressure difference between the inner sleeve and the outside world is as follows:

[0049]

[0050] Wherein, Q1 is the injection flow of the first through hole, R1 is the radius of the first through hole, ΔP is the pressure difference between the inner sleeve and the outside world, μ1 is the dynamic viscosity of the detection reagent, L1 is the sum of the lengths of the first through hole and the liquid inlet pipe. According to actual observation, Q1 will not generate bubbles and wall hanging when it is in the range of 0.3-1.0 mL / min, and uniform mixing of the sample liquid and detection reagent can be ensured when they are injected at the same time, so it is necessary to control the variables to ensure that Q1 does not exceed this range.

[0051]

[0052] Wherein, Q2 is the injection flow of the second through hole, R2 is the radius of the second through hole, ΔP is the pressure difference between the inner sleeve and the outside world, μ2 is the dynamic viscosity of the sample liquid, L2 is the sum of the lengths of the second through hole and the sample injection hole. Control Q2 in the range of 0.3-1.0 mL / min.

[0053] Assuming that the rotation of the inner sleeve is uniform, and the centers of the cross sections of the liquid inlet pipe and the first through hole are on the same horizontal plane, and the centers of the cross sections of the sample injection hole and the second through hole are on the same horizontal plane, the communication time lengths of the liquid inlet pipe and the first through hole and the sample injection hole and the second through hole are respectively:

[0054]

[0055] Wherein, t1 is the communication time length of the liquid inlet pipe and the first through hole, S1 is the sum of the inner diameters of the liquid inlet pipe and the first through hole, v1 is the linear velocity of the rotation of the inner sleeve.

[0056]

[0057] Wherein, t2 is the communication time length of the sample injection hole and the second through hole, S2 is the sum of the inner diameters of the sample injection hole and the second through hole, v1 is the linear velocity of the rotation of the inner sleeve.

[0058] The total injection volume V1 of the detection reagent is Q1*t1, and

[0059]

[0060] The total injection volume V2 of the sample liquid is Q2*t2, and

[0061]

[0062] Generally, V1 and V2 are a rated amount, which is set in the experimental design stage, when the batch of liquid reagents used for detection is the same, the relative change of μ1 and μ2 can be ignored, when the device-related parameters S1, S2, R1, R2, L1, L2 are unchanged, the following formula can be obtained Therefore, the ratio of the pressure difference between the inner sleeve and the outside and the rotation speed of the inner sleeve can accurately control the total injection amount of the sample liquid or the detection reagent.

[0063] After the injection is completed, the lifting plate is lifted to separate the cannula from the socket, the plug from the slot, and the bottom plate and the outer sleeve thereon are removed, and the reaction is carried out in the qPCR instrument, and whether the sample liquid contains the corresponding antibiotic resistance gene is obtained according to the reaction result.

[0064] The present application can be used for 256 antibiotic resistance genes of one sample, or 32 antibiotic resistance genes of 8 samples, or 16 antibiotic resistance genes of 16 samples, or 8 antibiotic resistance genes of 32 samples, or 4 antibiotic resistance genes of 64 samples for copy number quantitative detection (probe method) of experimental operation.

[0065] The present application can be used for 256 antibiotic resistance genes of one sample, or 32 antibiotic resistance genes of 8 samples, or 16 antibiotic resistance genes of 16 samples, or 8 antibiotic resistance genes of 32 samples, or 4 antibiotic resistance genes of 64 samples for copy number quantitative detection (probe method) of experimental operation. The TaqMan fluorescent probe is an oligonucleotide RNA probe, and the excitation fluorescent group is connected to the 5' end of the probe, and the quenched fluorescent group is at the 3' end. When PCR amplification is carried out, a specific fluorescent probe capable of binding to the template is added at the same time as a pair of primers, when the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenched group, and when PCR amplification is carried out, the 5'-3' exonuclease activity of Taq enzyme degrades the probe, so that the reporter fluorescent group and the quenched fluorescent group are separated, so that the fluorescent monitoring system can receive the fluorescent signal. The specific operation steps are as follows:

[0066] S1, extract the nucleic acid of the sample to be detected, confirm the sample type and concentration according to the sample detection report, if the sample needs to be diluted, dilute it with Nuclear-Free Water. The cDNA concentration of normal tissue is 10 ng / μL. The DNA sample concentration is diluted to 10 ng / μL;

[0067] S2, prepare the sample preMix according to the following table

[0068] Table 1 sample (including positive control) preMix preparation table

[0069]

[0070] The Mix of the positive control (PTC) is prepared by mixing plasmid standards of all antibiotic resistance genes (ARGs) in equal proportions, and the final concentration of each gene plasmid standard is 10-100 pM.

[0071] S3, prepare the sample Mix

[0072] Take a 96 or 384 well PCR plate and prepare the sample Mix according to the following table.

[0073] Table 2 sample (including positive control) Mix preparation table in PCR plate

[0074]

[0075] Each sample has at least 2 technical repeats, and the prepared sample Mix PCR plate is placed on ice or in a 4°C refrigerator for use.

[0076] S4, use the above detection sample adding device to perform automatic sample adding according to the Figures 7-11 sample array position information. After sample adding is completed, take out the 384 well plate and check if there is liquid on the upper surface. If there is liquid, cover it with a filter paper and press the filter paper with a paper stamp. Immediately take the filter paper and observe if there are liquid traces on the filter paper. If not, it means that the sample adding effect is good.

[0077] S5, fluorescence quantitative detection

[0078] Place the sample added 384 well plate in a 384 well qPCR instrument to perform qPCR reaction, set the amplification temperature program, set the 384 layout according to the 384 well plate array diagram, select all 5 fluorescence channels to collect fluorescence, select Taqman mode, and save the file.

[0079] Select the corresponding qPCR program Action, select "Probe-based expression analysis" in the probe method mode, select the corresponding program in the corresponding mode state Protocol drop-down list, and check the program temperature curve.

[0080] S6、Data processing

[0081] After running, check the Ct and SD of the same sample, remove some large deviation data, then process the image data, remove the abnormal data of the amplification curve, and then export the data results. After exporting the data, process the delivery data in the Excel table, which includes three tables of original, repeated data and data processing.

[0082] Absolute quantitative calculation:

[0083] If the absolute copy number of each gene needs to be calculated indirectly through the relative copy number, the 16s rRNA gene of the sample needs to be absolutely quantified, and then scaled according to the proportion. The scaling method is for reference only:

[0084] Objective gene absolute copy number = 16s rRNA gene absolute copy number * 2 -ΔC T

[0085] ΔC T = C T (gene) - C T (16s)

[0086] Note: C T (gene) and C T (16s) come from the same sample.

[0087] S7, Bioinformatics analysis and mapping

[0088] (1) Use qPCR experimental data to count the number, type, resistance mechanism and antibiotic type of ARGs, and draw a chart to show;

[0089] (2) Use heat map to show the abundance distribution pattern of ARGs in different samples. Use R package pheatmap to draw a cluster heat map. When only relative quantification is done, the mapping data uses Relative copy number = Copy (gene) / Copy (16S) , where Copy (gene) and Copy (16S) come from the same sample. Among them, Copy = 10 (31-C T ) / (10 / 3), the C T value of the off-machine data is null, and C TThe value is replaced with 31, so it is 1 when calculating Copy. When plotting, if the Copy value is 1, the Relative copy number is represented by "NA". Relative copy number * 10 6 A heatmap was plotted using logarithmic values ​​to the base 10. For absolute quantification, the data used in the plot was the absolute quantitative result, with null values ​​replaced by 1, and the heatmap was plotted using logarithmic values ​​to the base 10. The colors in the graph change from green to yellow to red, indicating a gradual increase in the abundance of antibiotic resistance genes. White indicates that the antibiotic resistance gene was not detected in the sample.

[0090] (3) Correlation analysis: Circos plots can visually show the types of antibiotics that ARGs detected in different samples are resistant to. Circos plots were drawn using the Perl Circos tool (version: 0.69-6).

Claims

1. An antibiotic resistance gene detection device, characterized by, The utility model relates to a kind of sample injection device, including rack (1) and detachably connect with the bottom plate (10) of rack (1), the bottom plate (10) is equipped with outer sleeve (2), outer sleeve (2) is equipped with sample groove (21) on the side wall, sample groove (21) bottom is equipped with sample injection hole (211), outer sleeve (2) bottom wall is further connected with liquid inlet pipe (41), the inner wall of outer sleeve (2) is rotatably connected with inner sleeve (3), the bottom of inner sleeve (3) is equipped with first through-hole (34) and second through-hole (35) that can be communicated with liquid inlet pipe (41) and sample injection hole (211) correspond, the top of inner sleeve (3) is equipped with upper cover (31), the top of rack (1) is equipped with lifting mechanism, and lifting mechanism is equipped with hollow assembly for manufacturing negative pressure environment in inner sleeve (3), the lower end of hollow assembly is detachably communicated with inner sleeve (3), and the lower portion of hollow assembly is equipped with rotating assembly for driving inner sleeve (3) rotation, and the lower end of rotating assembly is detachably connected with upper cover (31), and one side of rotating assembly is equipped with driving mechanism, and driving mechanism is equipped on hollow assembly; The hollow assembly includes a lifting plate (6) disposed on the lifting mechanism. The lifting plate (6) has an inner cavity (64) formed therein. The lifting plate (6) has a negative pressure pipe (63) in communication with the inner cavity (64). The lifting plate (6) has a hollow base (61) in communication with the inner cavity (64). The hollow base (61) has a cannula (62) connected thereto. The upper cover (31) has a socket (32) for receiving the cannula (62). The rotating assembly includes a gear (51) rotatably connected to the outer wall of the hollow base (61). The gear (51) has a plug strip (52) hung therefrom. The upper cover (31) has a slot (33) for receiving the lower end of the plug strip (52). The bottom of the lifting plate (6) is slidably connected to a rack (5) engaged with the gear (51). The driving mechanism is disposed at one end of the rack (5).

2. The antibiotic resistance gene detection device according to claim 1, wherein The driving mechanism includes a connecting strip (54) disposed at one end of the rack (5). The lifting plate (6) has a second electric cylinder (53). The second electric cylinder (53) has a telescopic rod fixedly connected to the connecting strip (54).

3. The antibiotic resistance gene detection device according to claim 1, wherein The lifting mechanism includes a first electric cylinder (11) disposed on the top of the rack (1). The lifting plate (6) is disposed at the lower end of the telescopic rod of the first electric cylinder (11). The lifting plate (6) has a guide rod (12) slidably connected to the top of the rack (1).

4. The antibiotic resistance gene detection device according to claim 1, wherein The liquid inlet pipe (41) is connected to a main pipe (4). The bottom of the rack (1) has a mounting groove (13). The bottom plate (10) is disposed in the mounting groove (13).

5. A method for detecting an antibiotic resistance gene for a non-disease diagnosis purpose, characterized by, The device is used for detection as follows: The device comprises a rack and a bottom plate detachably connected to the bottom of the rack, the bottom plate is provided with an outer sleeve, a sample groove is arranged on the side wall of the outer sleeve, a sample injection hole is arranged at the bottom of the sample groove, and a liquid inlet pipe is further communicated with the bottom of the side wall of the outer sleeve; an inner sleeve is sealingly and rotatably connected to the inner wall of the outer sleeve, the bottom of the inner sleeve is provided with a first through hole corresponding to the liquid inlet pipe and a second through hole corresponding to the sample injection hole; an upper cover is arranged at the top of the inner sleeve, a lifting mechanism is arranged at the top of the rack, a hollow assembly for creating a negative pressure environment in the inner sleeve is arranged on the lifting mechanism, the lower end of the hollow assembly is detachably communicated with the inner sleeve, a rotating assembly for driving the rotation of the inner sleeve is arranged at the lower part of the hollow assembly, the lower end of the rotating assembly is detachably connected with the upper cover, and a driving mechanism is arranged on one side of the rotating assembly. The hollow assembly comprises a lifting plate arranged on the lifting mechanism, an inner cavity is arranged in the lifting plate, a negative pressure pipe is arranged on the lifting plate and communicated with the inner cavity, a hollow base is arranged at the bottom of the lifting plate and communicated with the inner cavity, and a cannula is communicated with the lower end of the hollow base. The rotating assembly comprises a gear rotatably connected to the outer wall of the hollow base, and an insertion strip is hung on the gear, an insertion slot is arranged on the upper cover and used for inserting the lower end of the insertion strip, a rack is slidingly connected to the bottom of the lifting plate and engaged with the gear, and the driving mechanism is arranged at one end of the rack. The driving mechanism comprises a connecting strip arranged at one end of the rack, a second electric cylinder is arranged on the lifting plate, and the telescopic rod of the second electric cylinder is fixedly connected with the connecting strip. The lifting mechanism comprises a first electric cylinder arranged at the top of the rack, the lifting plate is arranged at the lower end of the telescopic rod of the first electric cylinder, and a guide rod is slidingly connected between the lifting plate and the top of the rack. A main pipe is communicated with the liquid inlet pipe, the bottom of the rack is provided with a mounting groove, and the bottom plate is arranged in the mounting groove. The detection method comprises the following steps: (1) In the initial state, ensure that the liquid inlet pipe is completely misaligned and closed with the first through hole, and the sample injection hole is completely misaligned and closed with the second through hole, connect the negative pressure pipe to the negative pressure pump, connect the liquid inlet pipe to the liquid storage tank, fill the detection reagent into the liquid inlet pipe, inject the sample liquid into the sample groove, and then place the bottom plate to ensure that the insertion hole is directly below the cannula and the insertion slot is directly below the insertion strip; (2) Lower the lifting plate until the cannula is inserted into the insertion hole and the insertion strip is inserted into the insertion slot, stop lowering the lifting plate, open the negative pressure pump, and suck the air in the inner sleeve through the cannula to control the formation of constant negative pressure in the inner sleeve; (3) Pull the rack to drive the gear and the insertion strip to rotate around the hollow base, the insertion strip drives the inner sleeve to rotate synchronously, when the liquid inlet pipe is communicated with the first through hole and / or the sample injection hole is communicated with the second through hole, the sample liquid and / or the detection reagent flows into the inner sleeve from the bottom of the outer sleeve and the inner sleeve to mix, continuously rotate the inner sleeve until the liquid inlet pipe is completely misaligned and closed with the first through hole and the sample injection hole is completely misaligned and closed with the second through hole, and the liquid injection operation is completed; (4) The ratio of the pressure difference between the inner sleeve and the outside world and the self-rotation speed of the inner sleeve is controlled to ensure that the rated amount of sample liquid and detection reagent is injected and liquid splashing and air bubble generation are avoided. (5) After the liquid injection is completed, the lifting plate is lifted to separate the insertion tube from the insertion hole, the insertion strip from the insertion slot, and the bottom plate and the outer sleeve thereon are removed and placed in the qPCR instrument for reaction, and whether the sample liquid contains the corresponding antibiotic resistance gene is determined according to the reaction result.

6. The method for detecting antibiotic resistance genes for non-disease diagnostic purposes according to claim 5, characterized in that, In step (1), the sample liquid is respectively injected into the corresponding sample groove according to the following array position distribution principle: S1 x A1, S1 x A2, S1 x A3... S1 x A m-2 , S1 x A m-1 , S1 x A m ; S2 x A1, S2 x A2, S2 x A3... S2 x A m-2 , S2 x A m-1 , S2 x A m ; S3 x A1, S3 x A2, S3 x A3... S3 x A m-2 , S3 x A m-1 , S3 x A m ;... S n-1 x A1, S n-1 x A2, S n-1 x A3... S n-1 x A m-2 , S n-1 x A m-1 , S n-1 x A m ; S n x A1, S n x A2, S n x A3... S n x A m-2 , S n x A m-1 , S n x A m ; or S1 x A1, S2 x A1, S3 x A1... S n-2 x A1, S n-1 x A1, S n x A1; S1 x A2, S2 x A2, S3 x A2... S n-2 x A2, S n-1 x A2, S n x A2; S1 x A3, S2 x A3, S3 x A3... S n-2 x A3, S n-1 x A3, S n x A3;... S1 x A m-1 , S2 x A m-1 , S3 x A m-1 ... S n-2 x A m-1 , S n-1 x A m-1 , S n x A m-1 ; S1 x A m , S2 x A m , S3 x A m ... S n-2 x A m , S n-1 x A m , S n x A m ; the order of S1 x A1, S2 x A1, S3 x A1... S Different sample liquids are sequentially injected into the corresponding sample grooves from top to bottom in the longitudinal direction and from left to right in the horizontal direction, wherein n is the number of samples to be detected, and m is the number of antibiotic resistance genes to be detected, and each antibiotic resistance gene of each sample is not less than 2 technical repeats.

7. The method for detecting antibiotic resistance genes for non-disease diagnostic purposes according to claim 6, characterized in that, One side of the sample liquid sample column is provided with a control column, and the control column contains positive and negative controls.

8. The method for detecting antibiotic resistance genes for non-disease diagnostic purposes according to claim 6, characterized in that, The sample liquid contains nucleic acid extracts of the samples to be detected and corresponding fluorescent probes of the antibiotic resistance genes to be detected.

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