CN115575483A - Direct target plate droplet growth device and using method thereof - Google Patents

Abstract

The invention discloses a direct target plate droplet growth device and a using method thereof, the direct target plate droplet growth device comprises: the base comprises a target plate bracket and an annular water tank, and the annular water tank surrounds the target plate bracket; the target plate is positioned in the target plate bracket, and one side surface of the target plate is provided with a plurality of hydrophobic rings; the sealing gasket is attached to one side face of the target plate and comprises a first water groove hole corresponding to the annular water tank and a plurality of first water drainage holes corresponding to the plurality of water drainage rings; the hole groove plate is positioned on the sealing gasket and comprises a second water groove hole corresponding to the first water groove hole and a plurality of second hydrophobic holes corresponding to the plurality of first hydrophobic holes, so that the annular water groove, the first water groove hole and the second water groove hole form the water groove holes, and the plurality of hydrophobic rings, the plurality of first hydrophobic holes and the plurality of second hydrophobic holes form the sample adding holes; and the top cover is positioned on the hole slot plate and is clamped with the base. The direct target plate micro-droplet growth device disclosed by the invention can shorten the time for identifying microorganisms and detecting drug sensitivity.

Description

Direct target plate droplet growth device and using method thereof

Technical Field

The invention relates to the technical field of detection, in particular to a direct target plate droplet growth device and a using method thereof.

Background

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a soft ionization mass spectrometry technology and has the characteristics of high speed, stability, sensitivity, accuracy, high resolution, low cost and the like. MALDI-TOF MS is introduced into a clinical microorganism laboratory and is mainly used for the rapid identification of bacteria and fungi at present. The identification principle is as follows: laser irradiates a cocrystal formed by a microbial sample and a matrix, the matrix enables biomolecules (mainly protein) contained in the microbial sample to be ionized after absorbing laser energy, charged biomolecules are subjected to energy obtaining, acceleration and focusing under the action of a high-voltage electric field, the time of the charged biomolecules reaching a TOF analyzer is in direct proportion to mass, a specific biomolecule fingerprint is formed by taking a mass-to-charge ratio (m/z) as an abscissa and an ion peak as an ordinate, and an identification result is matched by comparing the specific biomolecule fingerprint with a spectrum library. MALDI-TOF MS only needs 3-5 minutes for identifying the microorganisms, while the traditional biochemical identification card needs several hours to dozens of hours.

Whether infectious diseases can be treated timely and effectively depends on the identification speed of pathogenic microorganisms and is limited by the detection speed of drug sensitivity. Currently, the methods for detecting drug sensitivity of microorganisms mainly comprise: in vitro Antimicrobial Susceptibility Testing (AST), polymerase Chain Reaction (PCR), MALDI-TOF MS, and MALDI-TOF MS-based direct-on-target micro-droplet growth assay (DOT-MGA). AST is a reference method recommended by International organization for standardization (ISO), clinical and Laboratory Standards Institute (CLSI) and European Committee for antimicrobial drug sensitivity testing (EUCAST), tests the bactericidal or bacteriostatic ability of drugs in vitro, including diffusion, dilution, antibiotic concentration gradient, and automated instrumentation, and has the advantages of detecting unknown mechanism resistance, long time consumption, obtaining drug sensitivity results usually the next day, and no discrimination of microbial purity. PCR is used for rapidly detecting specific drug-resistant genes by using specific primers, and has the defects that unknown genes or unknown mechanism drug resistance cannot be detected, and the drug-resistant genes and drug-resistant phenotypes are sometimes inconsistent. MALDI-TOF MS drug sensitive detection is divided into three main categories. (1) Directly and rapidly detecting related biomolecules of a specific drug resistance mechanism, such as carbapenemase, beta-lactamase and the like, and has the defect that the drug resistance of an unknown mechanism cannot be detected. (2) Comparing the antibiotic MALDI-TOF MS fingerprint before and after incubation with the strain to be detected, the change of the fingerprint indicates the generation of hydrolase, and the method has the advantages of being capable of detecting various hydrolase resistances and having the defects of complicated operation steps and being incapable of detecting non-hydrolase resistances. (3) The DOT-MGA based on MALDI-TOF MS can complete the rapid identification of the microorganisms and the AST on one platform, has the advantages of the two, and avoids the defect that the traditional AST cannot distinguish the purity of the microorganisms to be detected.

In the prior art, the resistance of 24 Klebsiella pneumoniae and 24 Pseudomonas aeruginosa to meropenem is rapidly detected by using Rapid detection of anti-inflammatory resistance by MALDI-TOF mass spectrometry, which is a novel direct-on-target micro-droplet growth approach, namely Clin microbial infection, 2018 (7): 738-743, for the first time by using DOT-MGA based on MALDI-TOF MS. The specific operation steps are as follows:

(1) Fresh colonies were picked to prepare a suspension of the test bacteria at 0.5 McLeod turbidity, which was measured in CAMHB broth, 1:100 to 1X 10 ⁶ CFU/mL。

(2) Meropenem was dissolved in CAMHB broth and its concentration was adjusted to 4. Mu.g/mL.

(3) Mixing the diluted bacteria solution and meropenem solution in the same volume in a disposable MALDThe hydrophilic target ring of the I-TOF MS target plate forms a microdroplet. The final concentration of the bacterial liquid of the micro-drop in the drug sensitive ring is 5 multiplied by 10 ⁵ CFU/mL, the final concentration of meropenem solution is 2 mug/mL. Meanwhile, a growth control ring is arranged, namely the bacterial liquid after equal volume dilution and the CAMHB broth without the medicine, and the final concentration of the bacterial liquid of the micro-droplets is also 5 multiplied by 10 ⁵ CFU/mL. In the experiment, 5 different volumes of droplets were tested, including 2. Mu.L, 4. Mu.L, 6. Mu.L, 8. Mu.L and 10. Mu.L.

(4) The target plate was placed in an attached plastic transfer box (Brukton, inc.) with 4mL of water added to the bottom of the box, which served as a wet box to prevent evaporation of the microdroplets during incubation, incubated at 36 ℃ in air. Klebsiella pneumoniae, incubated for 3 hours, 4 hours and 18 hours; pseudomonas aeruginosa, incubated for 4 hours, 5 hours and 18 hours.

(5) Folding a low dusting wipe (kimtec Science, kimberly corporation, rosswal, usa) and "tapping" the droplet from the bottom side, the liquid was quickly removed by capillary effect to avoid interference of the broth components with the analysis. Subsequently, a normalized MALDI-TOF MS operation was performed.

(6) The microbial species within each target ring were identified using MALDI Biotyper 3.1 software (brueck dalton). In the growth control ring, the species identification is successful (the score is more than or equal to 1.7), and the test is effective. In the drug sensitive ring, the species identification is successful (the score is more than or equal to 1.7), and the strain is a drug-resistant strain; otherwise, the strain is a non-drug-resistant strain.

The results show that: (1) the microdroplets with the volume of 6 mu L reach the best determination performance for Klebsiella pneumoniae and pseudomonas aeruginosa; (2) under a microdroplet system of 6 mu L, incubating Klebsiella pneumoniae for 4 hours, wherein the detection rate in a growth control ring is 100%; (3) under a droplet system of 6 mu L, the detection rate of the pseudomonas aeruginosa after incubation for 4 hours is only 54.2 percent, and the detection rate of the pseudomonas aeruginosa after incubation for 5 hours is improved to 83.3 percent; (4) bacteria directly incubate and grow on a target plate, and remove broth and entrap thalli by using a low-dust wiping paper towel to lightly touch droplets, which is a necessary condition for ensuring the test to be effective; (5) transferring the target plate in a microdroplet form after the target plate is incubated outside the target plate, immediately removing the broth, and enabling the growth control ring not to be detected; after a short incubation time (15-60 min) again, the broth was removed and the detection rate in the growth control loop was proportional to the incubation time. For analytical reasons, the MALDI-TOF MS score of DOT-MGA is likely to be closely related to the amount of biofilm formation on the inner surface of the target circle, and not to the amount of bacteria suspended within the droplets.

Rapid and homogeneous testing of multiple antibiotics by the MALDI-TOF MS direct-on-target micro-porous assay, diagnostics,2021 (10): 1803, rapid batch detection of enterobacteriales for 24 common antibiotics by DOT-MGA based on MALDI-TOF MS, and the following improvements to the specific operating steps:

(1) Mixing 100 μ L of 5X 10 ⁵ CFU/mL of each bacterial solution to be tested was added to the wells of the microtiter plate containing the dry antibiotic and shaken at room temperature and 300rpm for 5 minutes to ensure complete dissolution of the antibiotic.

(2) Transferring 6 mu L of the bacteria liquid containing the antibiotics to be detected from each hole into a hydrophilic target ring of a disposable MALDI-TOF MS target plate to form microdroplets. At the same time, an equal volume of growth control loop without antibiotic was set.

(3) The target plate was placed in an attached plastic transfer box, with 4mL of water added to the bottom of the box, which served as a wet box to prevent evaporation of the droplets during incubation, and incubated in air at 36 ℃ for 6 hours, 8 hours, and 18 hours.

(4) After folding the low dusting wipe, the top "kiss" droplet, quickly removes liquid by capillary effect, avoiding broths components interfering with the analysis. Subsequently, a normalized MALDI-TOF MS operation was performed.

(5) In the growth control ring, the species identification is successful (the score is more than or equal to 1.7), and the test is effective. In the drug sensitive ring, the species identification is successful (the score is more than or equal to 1.7), and the strain is a drug-resistant strain; otherwise, the strain is a non-drug-resistant strain. And judging the sensitivity (S), the intermediary (I) or the drug resistance (R) of the strain to be detected according to the 1-2 concentration break points of each antibiotic.

The remaining 94 μ L of the antibiotic-containing bacterial suspension to be tested in each well of the microtiter plate was incubated at 36 ℃ for 18 hours in the air as a broth dilution method AST to evaluate the accuracy of DOT-MGA. The results show that: the detection rate in the control loop for growth at 6 hours and 8 hours in the system is 100%, but the detection rate in the control loop at 18 hours is only 75.2%. The authors noted that evaporation of water from the droplets interfered with growth of the bacteria to be tested and MALDI-TOF MS detection, probably because the simple plastic cartridges used in the experiments did not facilitate long-term humidity control. Thus, there is a need for an improved humidity control device.

In summary, the application prospect of DOT-MGA is very broad, but at present, there are still many disadvantages:

1. in the incubation process, the humidity is not well controlled, the water content of microdroplets is easy to evaporate, and the growth of bacteria to be detected and MALDI-TOF MS detection are interfered.

2. There is no physical separation between adjacent target rings, the droplet volume is limited, and adjacent droplets are miscible during handling and movement.

The broth removal method of 'light touch of low dust wiping paper towel' is very skillful and has the risk of cross contamination between adjacent target rings.

MALDI-TOF MS scoring of DOT-MGA was dependent on the amount of biofilm formation on the inner surface of the target circle.

5. High throughput and automated detection is difficult to achieve and requires further optimization and standardization.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the method solves the problems that in the incubation process, the water content of the droplets is easy to evaporate, the volume of the droplets is limited, the adjacent droplets are easy to mix and melt, the broth removing mode easily causes cross contamination between the adjacent target rings, and the method is not beneficial to high-throughput and automatic detection.

In order to solve the technical problems, the invention provides the following technical scheme:

a direct target plate droplet growth apparatus comprising:

a base including target plate brackets and an annular water trough surrounding the target plate brackets;

the target plate is positioned in the target plate bracket, and a plurality of hydrophobic rings are arranged on one side surface of the target plate;

the sealing gasket is attached to one side surfaces of the base and the target plate and comprises a plurality of first water groove holes corresponding to the annular water tank and a plurality of first water drainage holes corresponding to the plurality of water drainage rings;

the hole groove plate is positioned on the sealing gasket and comprises a plurality of second water groove holes corresponding to the first water groove holes and a plurality of second drainage holes corresponding to the first drainage holes, so that the annular water groove, the first water groove holes and the second water groove holes form the water groove holes, and the drainage rings, the first drainage holes and the second drainage holes form the sample adding holes;

the top cap is located on the hole frid, with the base block, and with the top of hole frid with the gap has all around of base, the air mediation of being convenient for.

The advantages are that: (1) Through setting up annular basin and top cap, can prevent effectively that droplet moisture from evaporating. (2) Through setting up sealed the pad, realize the physical separation between a plurality of adjacent hydrophobic rings on the used target plate, thoroughly solve the problem that adjacent droplet is easily mixed and melted. (3) Through the stack of sealed pad, hole frid, base and used target plate, form the application of sample hole, the application of sample volume of increase microdroplet is favorable to high flux, automatic application of sample and the liquid in each application of sample hole of siphoning off, make it can be under the condition of using or not using centrifugal equipment, at a plurality of hydrophobic intra-annular surfaces of used target plate, accelerate or form the fungus membrane naturally, avoid the cross contamination between a plurality of adjacent hydrophobic rings, shorten check time, improve work efficiency.

In an embodiment of the invention, the base further includes:

the annular water tank and the target plate bracket are positioned on the base plate and are not communicated;

a plurality of elastic keys positioned on the convex edge of the target plate bracket;

and one ends of the fixing columns are respectively fixed on the four corners of the base plate.

In an embodiment of the invention, the target plate comprises a target plate body, a plurality of the hydrophobic rings cover one side surface of the target plate body, and the number of the target plate body is the same as that of the target plate brackets.

In an embodiment of the present invention, the sealing pad further includes:

the sealing gasket body is penetrated by the first water groove holes and the first drain holes, and one side surface of the sealing gasket body is attached to the base and the surface of the target plate provided with the drain rings;

and the first sealing holes are respectively positioned at the four corners of the sealing gasket body.

In an embodiment of the present invention, the inner walls of the first hydrophobic holes are covered with a hydrophobic coating.

In an embodiment of the present invention, the orifice plate further includes:

the slotted plate body is penetrated by a plurality of second water slotted holes and a plurality of second hydrophobic holes;

and the second sealing holes are respectively positioned at the four corners of the orifice plate body.

In an embodiment of the invention, the inner walls of the second hydrophobic holes are covered with a hydrophobic coating.

In an embodiment of the invention, the direct target plate droplet growth apparatus further includes a plurality of fixing members, and after the other ends of the fixing posts sequentially pass through the first sealing hole and the second sealing hole, the fixing members are fixedly connected to the other ends of the fixing posts.

In an embodiment of the invention, the sealing pad is made of one of plastic, rubber or silica gel.

The invention also provides a use method of the direct target plate droplet growth device, which comprises the following steps:

placing the target plate into the target plate bracket;

placing the sealing gasket and the hole groove plate on the base in sequence and fixedly connecting the sealing gasket and the hole groove plate, so that the annular water groove, the plurality of first water groove holes and the plurality of second water groove holes form the water groove holes, and the plurality of hydrophobic rings, the plurality of first hydrophobic holes and the plurality of second hydrophobic holes form the sample adding holes;

adding a bacterial liquid of a bacterium to be detected into the sample adding hole;

adding a proper amount of sterile distilled water into the water tank hole;

clamping the top cover with the base for culture;

after the culture is finished, the thalli in the sample adding hole are centrifuged to the inner surface of the hydrophobic ring, and the formation of a bacterial membrane is accelerated;

and quickly absorbing the liquid in the sample adding hole, and performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry operation, analysis and identification and drug sensitivity results on the liquid.

Compared with the prior art, the invention has the beneficial effects that:

(1) The method can better control the detection condition, and thoroughly solve the problems that the moisture of the droplets is easy to evaporate, the volume of the droplets is limited, the adjacent droplets are easy to mix and melt, and the broth removal mode is not good, so that the cross contamination between the adjacent target rings is easy to cause.

(2) The target plate bracket, the first drain hole and the second drain hole can be correspondingly modified according to different specifications of the used target plate, so that the requirements of high flux and automatic detection are met.

(3) Further shortening incubation and detection time, being expected to thoroughly subvert the existing microorganism identification and drug sensitive system, providing more rapid and accurate detection information and helping clinicians to get a first chance in diagnosis and treatment of infectious diseases.

Drawings

FIG. 1 is a schematic view of a direct target plate droplet growth apparatus according to an embodiment of the present invention.

Fig. 2 is a schematic view of a base according to an embodiment of the invention.

FIG. 3 is a schematic view of a gasket according to an embodiment of the present invention.

Figure 4 is a flow chart of a method of using a direct target plate droplet growth apparatus according to an embodiment of the present invention.

Fig. 5 (a) and (B) are schematic diagrams illustrating identification of drug-resistant strains and sensitive strains of klebsiella pneumoniae according to an embodiment of the invention.

FIGS. 5 (C) and (D) are schematic diagrams illustrating identification of drug-resistant strains and sensitive strains of Escherichia coli according to an embodiment of the present invention.

FIGS. 5 (E) and (F) are schematic diagrams illustrating the identification of drug-resistant and susceptible strains of Pseudomonas aeruginosa according to embodiments of the present invention.

FIGS. 5 (G) and (H) are schematic diagrams illustrating the identification of drug-resistant and sensitive strains of Staphylococcus aureus according to an embodiment of the present invention.

Detailed Description

In order to facilitate the understanding of the technical solutions of the present invention for those skilled in the art, the technical solutions of the present invention will be further described with reference to the drawings attached to the specification.

The terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present application, "a plurality" means two or more unless specifically limited otherwise.

Referring to fig. 1, the present invention provides a direct target plate droplet growth apparatus, which includes a base 100, a target plate 200, a sealing gasket 300, a well plate 400, and a top cover 500. Therein, the base 100 comprises target plate brackets 120 and an annular water trough 130, the annular water trough 120 surrounding the target plate brackets 130. The target board 200 is positioned in the target board bracket 120, and a plurality of hydrophobic rings 210 are disposed on one side surface of the target board 200. The gasket 300 is attached to one side of the base 100 and the target plate 200, and the gasket 300 includes a plurality of first water groove holes 310 corresponding to the annular water tank 130 and a plurality of first water drain holes 320 corresponding to the plurality of water drain rings 210. The orifice plate 400 is disposed on the gasket 300 and includes a second water slot 410 corresponding to the plurality of first water slots 310 and a plurality of second water drainage holes 420 corresponding to the plurality of first water drainage holes 320, such that the annular water tank 130, the plurality of first water slots 310 and the plurality of second water slots 410 form water slot holes, and the plurality of water drainage rings 210, the plurality of first water drainage holes 320 and the plurality of second water drainage holes 420 form sample addition holes. The top cover 500 is disposed on the hole groove plate 400, engaged with the base 100, and has a gap with the top of the hole groove plate 400 and the periphery of the base 100, so as to facilitate air circulation.

Referring to fig. 1 and 2, in an embodiment of the present invention, the base 100 further includes a base plate 130, a plurality of elastic buttons 140 and a fixing post 150. Wherein the target plate brackets 120 and the annular water trough 130 are located on the base plate 130, and the target plate brackets 120 are not in communication with the annular water trough 130. The plurality of elastic keys 140 are positioned on the convex edge of the target plate bracket 120, and in a normal state, the plurality of elastic keys 140 and the convex edge of the target plate bracket 120 are at the same horizontal position, when the target plate 200 needs to be replaced, the keys 140 are pressed down, and the horizontal position of the keys 140 is lower than that of the convex edge of the target plate bracket 120, so that the target plate replacing 200 has a better acting point, and the target plate 200 can be conveniently taken out of the target plate bracket 120. When the replacement of a new target board 200 is completed, the resilient keys 140 thereof automatically return to the same horizontal plane as the raised edges of the target board brackets 120. Wherein, after the target board 200 is clamped on the target board bracket 120, it is at the same level with the flange of the target board bracket 120. The number of the fixing posts 150 is plural, and in this embodiment, 4, and the fixing posts are respectively fixed on four corners of the base plate 130. There is no limitation on the size of the base plate 130, nor on the number of target plate brackets 120, and the securing posts 150 are, for example, threaded studs. In this embodiment, the number of target plate brackets 120 is 2.

Referring to fig. 1, in an embodiment of the invention, the target board 200 further includes a target board body 220, and a plurality of hydrophobic rings 210 are covered on one side surface of the target board body 220. The target plate body 220 is positioned in the target plate bracket 120, the shape of the target plate body is the same as that of the target plate bracket 120, the number of the target plate bodies 220 is the same as that of the target plate brackets 120, but the number of the hydrophobic rings 210 on each target plate body 220 is variable and is determined according to the size of the target plate body 220.

Referring to fig. 1 and 3, in an embodiment of the present invention, the gasket 300 further includes a gasket body 330 and a first sealing hole 340, and the plurality of first water groove holes 310 and the plurality of first water drainage holes 320 penetrate through the gasket body 330. When the target board 200 is engaged with the target board bracket 120, one side of the gasket 300 is engaged with the base 100 and the target board 200, and the first water slots 310 correspond to the annular water groove 130, so that distilled water can enter the annular water groove 120 when the distilled water is poured into the first water slots 310. The first hydrophobic holes 320 correspond to the hydrophobic rings 210, and when the bacteria solution of the bacteria to be tested is dripped into the first hydrophobic holes 320, the bacteria solution can enter the inner surfaces of the hydrophobic rings 210. The number of the first sealing holes 340 is a plurality, in this embodiment 4, and the first sealing holes are respectively located at four corners of the gasket body 330. Wherein the inner walls of the first plurality of hydrophobic holes 320 are covered with a hydrophobic coating. The material of the sealing pad 300 is a natural or artificial synthetic material with certain elasticity, such as plastic, rubber or silica gel in this embodiment, and the physical separation is formed between the adjacent hydrophobic rings 210 by adhering to the side surface of the target plate 200 with the plurality of hydrophobic rings 210, so as to thoroughly solve the problems of limited droplet volume, mixing and pollution.

Referring to fig. 1, in an embodiment of the present invention, the well plate 400 further includes a well plate body 430 and a second sealing hole 440, wherein a plurality of second water well holes 410 and a plurality of second hydrophobic holes 420 penetrate the well plate body 430. The plate body 430 is attached to the other side of the gasket 300, and the second water grooves 410 correspond to the annular water tank 110 and the first water grooves 310, and the second water draining holes 420 correspond to the draining rings 210 and the first water draining holes 320. The number of the second sealing holes 440 is a plurality, in this embodiment 4, and the second sealing holes are respectively located at four corners of the orifice plate body 430. The direct target plate droplet growth device further comprises a fixing piece 450, and when the direct target plate droplet growth device is used, one end of the fixing column 150 sequentially penetrates through the first sealing hole 340 and the second sealing hole 440, the fixing piece 450 is sleeved on the fixing column 150, the base 100, the target plate 200, the sealing gasket 300 and the hole groove plate 400 are fixedly connected, at the moment, a plurality of water groove holes communicated with the bottom are formed by the second water groove holes 410, the annular water groove 110 and the first water groove holes 310, and when the direct target plate droplet growth device is used, a proper amount of sterile distilled water is added into the water groove holes, so that the humidity of the internal environment of the direct target plate droplet growth device is kept. A plurality of second hydrophobic holes 420 and a plurality of hydrophobic rings 210 and a plurality of first hydrophobic holes 320 form a plurality of application holes, through the stack of a plurality of hydrophobic holes, make application hole have hydrophobic inner wall and certain degree of depth, after the fungus liquid that awaits measuring of great volume adds application hole, even not with the help of centrifugal equipment, also can directly form the fungus membrane at the internal surface of hydrophobic rings 210, shorten check-out time. Wherein the inner walls of the first hydrophobic holes 320 and the second hydrophobic holes 420 are covered with a hydrophobic coating to facilitate the formation of a pellicle on the hydrophobic ring 210, and the fixing member 450 is, in this embodiment, a nut. Top cap 500 one side indent, with base 100 block, and with the top of orifice frid 400 and base 100 all around have the gap, the air mediation of being convenient for ensures the demand of cultivation in-process thalli to oxygen.

Referring to fig. 1 and 4, the present invention also provides a method of direct target plate droplet growth apparatus, comprising the steps of:

s100, placing the target plate into the target plate bracket.

S200, will sealed pad with the hole frid is placed in proper order on the base and fixed connection, makes annular water tank, a plurality of first water slot hole and a plurality of second water slot hole constitute the water slot hole makes it a plurality of hydrophobic ring, a plurality of first hydrophobic hole and a plurality of the hydrophobic hole of second constitutes the application of sample hole.

The base 100, the target plate 200, the gasket 300 and the slot plate 400 are fixedly connected by the fixing posts 150 and the fixing members 450 to form a whole.

S300, adding the bacterial liquid of the bacteria to be detected into the sample adding hole.

Wherein the bacterial liquid of the bacteria to be tested contains or does not contain antibiotics. The sample adding holes can be classified into drug sensitive holes or growth control holes, and the concentration of antibiotics is set according to drug sensitive requirements.

S400, adding a proper amount of sterile distilled water into the water tank hole.

Wherein, the added distilled water does not overflow the water slot hole.

And S500, clamping the top cover with the base for culturing.

Wherein, the device should be cultured in an incubator for a required period of time.

S600, after the culture is finished, the thalli in the sample adding hole are centrifuged to the inner surface of the hydrophobic ring, and the formation of a bacterial membrane is accelerated.

Wherein, the thalli in the sample adding hole is centrifuged to the surface of the target plate 200 in the hydrophobic ring 210 by a flat horizontal centrifuge to form a bacterial membrane; or directly forming a bacterial film on the surface of the target plate 200 in the hydrophobic ring 210 by using the larger volume of the bacterial liquid to be detected in the sample adding hole and the adhesion growth characteristic of bacteria without centrifugation, thereby shortening the detection time.

S700, quickly absorbing the liquid in the sample adding hole, and carrying out matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the liquid, and analyzing and identifying and drug sensitivity results.

Wherein, can use the pipettor through the automation instrument or manual, inhale the fungus liquid in the sample loading hole fast, avoid the meat and liquid composition to disturb the analysis.

Referring to fig. 5 (a) to 5 (H), in an embodiment of the present invention, fig. 5 (a) and (B) are schematic diagrams illustrating identification of resistant strains and sensitive strains of klebsiella pneumoniae, fig. 5 (C) and (D) are schematic diagrams illustrating identification of resistant strains and sensitive strains of escherichia coli, fig. 5 (E) and (F) are schematic diagrams illustrating identification of resistant strains and sensitive strains of pseudomonas aeruginosa, and fig. 5 (G) and (H) are schematic diagrams illustrating identification of resistant strains and sensitive strains of staphylococcus aureus. And (4) identifying the microbial species in each sample adding hole, growing in a control hole, successfully identifying the species (the score is more than or equal to 1.7), and effectively testing. In the drug sensitive hole, the species identification is successful (the score is more than or equal to 1.7), and the drug resistant strain is obtained; otherwise, the strain is a non-drug resistant strain.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes 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, and any reference signs in the claims are not to be construed as limiting the claims.

The above-mentioned embodiments only represent embodiments of the present invention, and the scope of the present invention is not limited to the above-mentioned embodiments, and it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit of the present invention, and these embodiments are all within the scope of the present invention.

Claims (10)

1. A direct target plate droplet growth apparatus, comprising:

a base including target plate brackets and an annular water trough surrounding the target plate brackets;

the target plate is positioned in the target plate bracket, and one side surface of the target plate is provided with a plurality of hydrophobic rings;

the sealing gasket is attached to one side surfaces of the base and the target plate and comprises a plurality of first water groove holes corresponding to the annular water tank and a plurality of first water drainage holes corresponding to the plurality of water drainage rings;

the hole groove plate is positioned on the sealing gasket and comprises a plurality of second water groove holes corresponding to the first water groove holes and a plurality of second drainage holes corresponding to the first drainage holes, so that the annular water groove, the first water groove holes and the second water groove holes form the water groove holes, and the drainage rings, the first drainage holes and the second drainage holes form the sample adding holes;

the top cap is located on the hole frid, with the base block, and with the top of hole frid with the gap has all around of base, the air mediation of being convenient for.

2. The direct target plate droplet growth apparatus of claim 1, wherein the base further comprises:

the annular water tank and the target plate bracket are positioned on the base plate and are not communicated;

a plurality of elastic keys positioned on the convex edge of the target plate bracket;

and one end of each fixing column is fixed at the four corners of the base plate.

3. The direct target plate droplet growth apparatus of claim 1, wherein the target plate comprises a target plate body, a plurality of the hydrophobic rings cover one side surface of the target plate body, and the number of the target plate body is the same as the number of the target plate brackets.

4. The direct target plate droplet growth apparatus of claim 2, wherein the gasket further comprises:

the sealing gasket body is penetrated by the first water groove holes and the first water drainage holes, and one side surface of the sealing gasket body is attached to the base and the surface of the target plate provided with the drainage rings;

and the first sealing holes are respectively positioned at the four corners of the sealing pad body.

5. The direct target plate droplet growth apparatus of claim 4, wherein the inner walls of the first plurality of hydrophobic wells are coated with a hydrophobic coating.

6. The direct target plate droplet growth apparatus of claim 4, wherein the well plate further comprises:

the slotted plate body is penetrated by a plurality of second water slotted holes and a plurality of second hydrophobic holes;

and the second sealing holes are respectively positioned at the four corners of the orifice plate body.

7. The direct target plate droplet growth apparatus of claim 6, wherein a plurality of the second hydrophobic wells are coated on an inner wall with a hydrophobic coating.

8. The direct target plate droplet growth apparatus of claim 6, further comprising a plurality of fixtures, wherein the fixtures are fixedly connected to the other ends of the fixing posts after the other ends of the fixing posts pass through the first sealing hole and the second sealing hole in sequence.

9. The direct target plate droplet growth apparatus of claim 1, wherein the gasket is made of one of plastic, rubber, or silicone.

10. Use of a direct target plate droplet growth apparatus according to any of claims 1 to 9, comprising the steps of:

placing the target board into the target board bracket;

placing the sealing gasket and the hole groove plate on the base in sequence and fixedly connecting the sealing gasket and the hole groove plate, so that the annular water groove, the plurality of first water groove holes and the plurality of second water groove holes form the water groove holes, and the plurality of hydrophobic rings, the plurality of first hydrophobic holes and the plurality of second hydrophobic holes form the sample adding holes;

adding a bacterial liquid of the bacteria to be detected into the sample adding hole;

adding a proper amount of sterile distilled water into the water tank hole;

clamping the top cover with the base for culturing;

after the culture is finished, the thalli in the sample adding hole are centrifuged to the inner surface of the hydrophobic ring, and a bacterial membrane is formed in an accelerated manner;

and quickly absorbing the liquid in the sample adding hole, and performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry operation, analysis and identification and drug sensitivity results on the liquid.

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