A rapid microorganism detection reaction container and a rapid microorganism detection device

Abstract

The utility model provides a kind of microbial rapid detection reaction container, including container main body, conical bottom, drain pipe, sealing cover, medicine injection tube, liquid injection rubber plug and filter membrane;Conical bottom is fixed in container main body bottom end, to form the closed reaction cavity inside container main body, drain pipe is fixed in conical bottom bottom end, and sealing cover is detachably installed in the end of drain pipe away from conical bottom;Medicine injection tube is fixedly installed in container main body top surface, and liquid injection rubber plug is fixedly installed in the end of medicine injection tube away from container main body, and filter membrane is fixed in the end of reaction cavity close to conical bottom.And by puncturing needle punctures liquid injection rubber plug, sample or reaction reagent is injected into reaction cavity, and reaction cavity is not directly contacted with external environment;Reaction cavity is in sealed state, satisfies aseptic operation requirement.Sample solution can be filtered by filter membrane to sample solution, only retain microorganism, prevent other components in sample solution from interfering with detection process, improve the detection efficiency and accuracy of microorganism.

Description

Technical Field

[0001] This utility model relates to the field of microbial detection technology, specifically to a rapid microbial detection reaction container and a rapid microbial detection device. Background Technology

[0002] In the fields of food, pharmaceuticals, pesticides, soil, and environment, there are numerous studies on microbial contamination and related standards and methods. Currently, microbial detection is mainly carried out through laboratory culture methods or ATP bioluminescence methods. Culture methods primarily involve isothermal and timed cultivation of microorganisms, followed by visual counting of colonies. The disadvantage of this method is its long detection time. ATP bioluminescence technology not only relies on the ATP produced by microorganisms during growth but also adds an excess of ADP. The adenosine kinase within the microorganisms converts ADP into ATP, and the fluorescence intensity (relative light units, RLU) reflects the ATP content in the sample, thereby improving detection sensitivity, amplifying the bioluminescent signal, and ultimately determining the presence of microbial growth, significantly shortening the detection time.

[0003] Currently, in the rapid detection of microorganisms using ATP bioluminescence, EP tubes are often used directly as the reaction vessel due to the lack of dedicated rapid microbial detection reaction containers. Microbial detection is a sterile procedure requiring strict environmental conditions and aseptic operation. However, using EP tubes as reaction containers necessitates frequent opening of the cap to inject sample solutions, ADP substrate reagents, luciferase, cell lysis agents, etc. This process exposes the reaction chamber of the EP tube to direct contact with the external environment, easily leading to reaction solution contamination and failing to meet aseptic requirements. Furthermore, EP tubes cannot filter the sample solution to retain only microorganisms, which may allow other components in the sample solution (such as antibiotics, preservatives, heavy metal ions, or other substances that interfere with the ATP bioluminescence reaction) to interfere with the detection process, reducing the detection efficiency and accuracy of microorganisms. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid microbial detection reaction vessel that can filter the sample solution to retain only microorganisms and meet aseptic operation requirements when performing rapid microbial detection using the ATP bioluminescence method, thereby solving the problems existing in the background art.

[0005] This utility model adopts the following technical solution: a rapid microbial detection reaction container, including a container body, a conical bottom, a drain pipe, a sealing cap, a drug injection pipe, a liquid injection stopper, and a filter membrane; the container body has a hollow structure, the conical bottom is fixed to the bottom end of the container body, so that the inside of the container body forms a closed reaction chamber, the drain pipe is fixed to the bottom end of the conical bottom, and the sealing cap is detachably installed at the end of the drain pipe away from the conical bottom;

[0006] At least one of the drug injection tubes is fixedly installed on the top surface of the container body, and the liquid injection stopper is fixedly installed at the end of the drug injection tube away from the container body. The filter membrane is fixedly installed in the reaction chamber near the conical bottom.

[0007] Furthermore, the rapid microbial detection reaction container also includes a base, which is fixedly connected to the bottom of the container body and integrally formed with the container body. The base has a cylindrical structure, and the drain pipe and the sealing cap installed on the drain pipe are located in the inner cavity of the base.

[0008] Furthermore, the rapid microbial detection reaction container also includes an air inlet pipe, which is fixedly installed on the top surface of the container body, and the sealing cap is detachably installed at the end of the air inlet pipe away from the container body.

[0009] Furthermore, the sealing cap includes a sealing part and a handle part. The sealing part has a columnar structure and is used to fit inside the drain pipe or air inlet pipe. The handle part is fixedly installed at one end of the sealing part, and the outer diameter of the handle part is larger than the outer diameter of the sealing part.

[0010] Furthermore, a support ring is fixedly provided at one end of the reaction chamber near the conical bottom, and the filter membrane is disposed on the top surface of the support ring. The filter membrane is fixedly installed on the support ring by a fixing ring.

[0011] Furthermore, the container body has a quadrangular prism structure, with one set of opposite sides being smooth and the other set of opposite sides being rough.

[0012] Furthermore, the injection stopper is a butyl rubber stopper; the pore size of the filter membrane is between 0.22μm and 0.45μm.

[0013] Furthermore, a rapid microbial detection device includes a liquid injection assembly, a fluorescence spectrometer, and a rapid microbial detection reaction container; the liquid injection assembly is used to extract samples or reaction reagents and puncture the liquid injection stopper to inject the samples or reaction reagents into the reaction chamber; the fluorescence spectrometer is used to measure the fluorescence intensity of the solution in the reaction chamber and to quantify the total amount of microorganisms in the sample.

[0014] Furthermore, the rapid microbial detection device also includes a gas injection assembly, which is sealed to the air inlet tube to inject sterile gas into the reaction chamber; the liquid injection assembly includes a disposable syringe; the gas injection assembly includes a disposable syringe or a micro air pump, and the air inlet tube can be sealed to the nipple of the disposable syringe or the air tube of the micro air pump.

[0015] The rapid microbial detection reaction container of this invention allows for sample or reaction reagent injection into the reaction chamber via a puncture needle piercing the injection stopper during the injection operation. The reaction chamber remains in direct contact with the external environment. Simultaneously, the reaction chamber remains sealed during the sample-reaction reaction, minimizing contamination of the reaction solution and meeting aseptic operation requirements. After the sample solution is injected into the reaction chamber, it is filtered through a membrane to retain only microorganisms. This prevents interference from other components in the sample solution (such as antibiotics, preservatives, heavy metal ions, or other substances that interfere with the ATP luminescence reaction) and improves the detection efficiency and accuracy of microorganisms. Attached Figure Description

[0016] Appendix Figure 1 This is a schematic diagram of the structure of the rapid microbial detection reaction vessel of this utility model.

[0017] Appendix Figure 2 This is a cross-sectional structural diagram of the rapid microbial detection reaction vessel of this utility model.

[0018] Appendix Figure 3 This is an appendix to the utility model Figure 2 A magnified structural diagram of part A in the middle.

[0019] Appendix Figure 4 This is a schematic diagram of the structure of the sealing cap 5 in this utility model.

[0020] The reference numerals in the attached drawings are explained as follows: Container body 1, Reaction chamber 101, Smooth surface 102, Textured surface 103, Base 2, Conical bottom 3, Drain pipe 4, Sealing cap 5, Sealing part 51, Handheld part 52, Injection tube 6, Injection rubber stopper 7, Air inlet pipe 8, Support ring 9, Filter membrane 10, Fixing ring 11. Detailed Implementation

[0021] To more clearly illustrate the technical solutions in the embodiments of this utility model or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. The invention will be further described below in conjunction with the drawings:

[0022] Example 1

[0023] Refer to the instruction manual appendix Figure 1 , 2 A rapid microbial detection reaction container includes a container body 1, a base 2, a conical bottom 3, a drain pipe 4, a sealing cap 5, a drug injection pipe 6, a liquid injection rubber stopper 7, an air inlet pipe 8, a support ring 9, a filter membrane 10, and a fixing ring 11.

[0024] Both the container body 1 and the base 2 are quadrangular prism structures. The container body 1 is a hollow structure, and the conical base 3 is fixedly connected to the bottom end of the container body 1, so that a closed reaction chamber 101 is formed inside the container body 1. The base 2 is fixedly connected to the bottom end of the container body 1 and is integrally formed with the container body 1. The base 2 is a hollow cylindrical structure with an open bottom surface.

[0025] Two injection tubes 6 are fixedly installed on the top surface of the container body 1. A liquid injection stopper 7 is fixedly installed at the end of the injection tube 6 furthest from the container body 1. One injection tube 6 is used to inject the sample solution, and the other injection tube 6 is used to inject the reaction reagent (such as ADP, luciferase, cell lysis agent, etc.). The liquid injection stopper 7 is made of butyl rubber. Butyl rubber stoppers facilitate needle puncture and liquid injection, and after the needle is withdrawn, they can close the puncture hole due to their own elasticity, ensuring that the reaction chamber 101 is sealed. Furthermore, the butyl rubber stopper can be punctured multiple times while maintaining its sealed state.

[0026] To filter the sample solution and retain only microorganisms, preventing interference from other components in the sample solution (such as antibiotics, preservatives, heavy metal ions, or other substances that interfere with the ATP luminescence reaction) and improving the detection efficiency and accuracy of microorganisms, a filter membrane 10 is fixedly installed at one end of the reaction chamber 101 near the conical bottom 3. The filter membrane 10 is used to filter the sample solution and retain microorganisms. The filter membrane 10 is selected from cellulose membranes, polyamide membranes, or polyethersulfone membranes depending on the specific application, and its pore size is between 0.22 μm and 0.45 μm.

[0027] Refer to the instruction manual appendix Figure 2 , 3 Specifically, the method for fixing the filter membrane 10 in the reaction chamber 101 is as follows: a support ring 9 is fixedly provided at one end of the reaction chamber 101 near the conical bottom 3. The support ring 9 is fixedly connected to the inner wall of the container body 1. The filter membrane 10 is disposed on the top surface of the support ring 9. A fixing ring 11 is provided on the top surface of the filter membrane 10. The fixing ring 11 is matched and sleeved with the container body 1. The filter membrane 10 is clamped and fixed by the fixing ring 11 and the support ring 9.

[0028] The drain pipe 4 is fixed at the bottom of the conical base 3, and the sealing cap 5 is detachably installed at the end of the drain pipe 4 away from the conical base 3. The drain pipe 4 and the sealing cap 5 installed on the drain pipe 4 are located in the inner cavity of the base 2. The conical base 3 and the drain pipe 4 facilitate the discharge of the filtered sample solution in the reaction chamber 101, and the sealing cap 5 facilitates the sealing of the drain pipe 4. After the filtered sample solution is discharged, the sealed environment in the reaction chamber 101 is maintained.

[0029] The inventors discovered that after the sample solution is injected into the reaction chamber 101, since there is only one channel, the drain pipe 4, in order to enable the sample solution to pass through the filter membrane 10 quickly and complete the filtration, an air inlet pipe 8 is fixedly installed on the top surface of the container body 1, and a sealing cap 5 is detachably installed at the end of the air inlet pipe 8 away from the container body 1.

[0030] In one embodiment, the filtration of the sample solution is performed inside a biosafety cabinet to ensure that sterile gas enters the reaction chamber 101.

[0031] To accelerate filtration efficiency and rate, a gas injection assembly is connected to the air inlet pipe 8. This assembly includes a disposable syringe or a miniature air pump. The nozzle of the disposable syringe or the tubing of the miniature air pump can be sealed to the air inlet pipe 8. Sterile gas is injected into the reaction chamber 101 via the disposable syringe or miniature air pump to accelerate the filtration efficiency and rate of the sample solution. After the sample solution is filtered, two sealing caps 5 are fitted onto the air inlet pipe 8 and the drain pipe 4 to maintain a sealed environment within the reaction chamber 101.

[0032] In another embodiment, when the filtration operation is not performed within the biosafety cabinet, an injection assembly is connected to the air inlet pipe 8. This assembly includes a disposable syringe or miniature air pump and a bacterial filter. The air inlet pipe 8 can be sealed to the bacterial filter. The bacterial filter comprises a filter medium and a plastically molded bottom cover and top cover. The bottom and top covers, when snapped together, form a cavity structure. The filter medium is disposed within this cavity structure. An air outlet and an air inlet, communicating with the cavity structure, are respectively connected to the bottom and top covers. The filter medium is a filter element or membrane used for filtering microorganisms. The air outlet can be sealed to the air inlet pipe 8. The filter element or membrane of the bacterial filter can filter airborne microorganisms, preventing them from entering the reaction chamber 101 and interfering with the detection process, as well as affecting the accuracy of microbial detection.

[0033] To accelerate filtration efficiency and rate, the tip of the disposable syringe or the tubing of the micro-pump is sealed to the inlet of the bacterial filter, and the outlet of the bacterial filter is sealed to the inlet pipe 8. Sterile gas is injected into the reaction chamber 101 via the disposable syringe or micro-pump to expedite the filtration of the sample solution. After filtration, the bacterial filter is removed, and the two sealing caps 5 are fitted onto the inlet pipe 8 and the outlet pipe 4 to maintain a sealed environment within the reaction chamber 101.

[0034] Refer to the instruction manual appendix Figure 4The sealing cap 5 includes a sealing part 51 and a handle part 52. The sealing part 51 has a columnar structure and is used to fit inside the drain pipe 4 or the air inlet pipe 8 to seal the drain pipe 4 or the air inlet pipe 8. The handle part 52 is fixedly installed on one end of the sealing part 51, and the outer diameter of the handle part 52 is larger than the outer diameter of the sealing part 51. The handle part 52 facilitates the installation and removal of the sealing part 51 from the drain pipe 4 or the air inlet pipe 8.

[0035] To improve the accuracy of the fluorescence intensity measurement of the solvent in the reaction chamber 101 by the fluorometer, one set of opposite sides of the container body 1 is smooth 102 and the other set of opposite sides is rough 103; for example, one set of opposite sides is frosted glass and the other set of opposite sides is transparent glass, or one set of opposite sides is made of opaque material and the other set of opposite sides is made of transparent material.

[0036] When measuring fluorescence intensity with a fluorophotometer, the two light surfaces 102 of the container body 1 are parallel and the distance between them is fixed. This ensures that the optical path remains consistent throughout the entire detection process when the incident light passes perpendicularly through the solvent in the reaction chamber 101, thus guaranteeing the accuracy of the fluorescence intensity. At the same time, the two light surfaces 102 of the container body 1 are planar and perpendicular to the light surface 102, which ensures that the light passes through the solvent in the reaction chamber 101 in a straight line, reducing refraction or scattering caused by interface irregularities. This improves the accuracy of the fluorophotometer in measuring the fluorescence intensity of the solvent in the reaction chamber 101.

[0037] In this embodiment, the method of using the rapid microbial detection reaction vessel is as follows: A target volume of sample solution is drawn using a disposable syringe, and the syringe needle is inserted into the injection stopper 7 on the first injection tube 6 to inject the entire sample solution into the reaction chamber 101. The needle is then removed. The sealing cap 5 on the air inlet tube 8 is removed, and a bacterial filter is sealed and connected to the air inlet tube 8. The gas injection assembly is sealed and connected to the air inlet end of the bacterial filter. The sealing cap 5 on the drain tube 4 is then removed, and sterile gas is injected into the reaction chamber 101 through the gas injection assembly. The sample solution is filtered through the filter membrane 10 and discharged from the drain tube 4. Microorganisms remain on the filter membrane 10. After filtration, the bacterial filter is removed from the air inlet tube 8, and the sealing cap 5 is reinstalled on the air inlet tube 8 and the drain tube 4.

[0038] Use a disposable syringe to draw the target volume of cell lysis agent, and puncture the injection stopper 7 on the second injection tube 6 through its puncture needle to inject all the cell lysis agent into the reaction chamber 101. Remove the puncture needle and shake the microbial rapid detection reaction container (a reciprocating shaker can be selected) to lyse the microbial cells and release the AK in the microorganisms.

[0039] A target volume of ADP substrate reagent is drawn using a disposable syringe, and the syringe needle is inserted into the injection stopper 7 on the second injection tube 6 to inject all the ADP substrate reagent into the reaction chamber 101. The needle is then removed, and the rapid microbial detection reaction container (a reciprocating shaker can be selected) is shaken to catalyze the generation of ATP from the exogenously added ADP substrate reagent, thereby increasing the ATP content.

[0040] Finally, a target volume of luciferase and luciferin is drawn using a disposable syringe, and the syringe needle is inserted into the injection stopper 7 on the second injection tube 6 to inject all of the luciferase and luciferin into the reaction chamber 101. The needle is then removed, and the microbial rapid detection reaction container (a reciprocating shaker can be selected) is shaken to convert the energy generated during the breaking of ATP high-energy bonds into a biofluorescent signal.

[0041] Subsequently, the rapid microbial detection reaction vessel can be tested using a fluorescence spectrophotometer, and the presence of microorganisms in the sample can be determined based on the intensity of the biofluorescence signal.

[0042] Example 2

[0043] A rapid microbial detection device includes a liquid injection assembly, a fluorescence spectrometer, a gas injection assembly, and the rapid microbial detection reaction container in Example 1.

[0044] The injection assembly uses a disposable syringe to draw samples or reaction reagents, and injects the samples or reaction reagents into the reaction chamber 101 by puncturing the injection stopper 7 with a puncture needle.

[0045] A fluorophotometer consists of a main body and a cover. The main body has a detection chamber for mounting a rapid microbial detection reaction container. When the cover is closed, the detection chamber becomes a dark chamber. The detector inside the main body measures the fluorescence intensity of the solution within the reaction chamber 101, indirectly quantifying the total amount of microorganisms in the sample. Fluorophotometers (also known as fluorescence spectrophotometers) are now standard products and are readily available on the market. Companies such as Shanghai Lingguang, Tianjin Tuopu, and Shanghai Sanke all manufacture such products, so further details are omitted.

[0046] The gas injection assembly is used to seal and connect to the air inlet pipe 8 or the bacterial filter of the air inlet pipe 8, injecting sterile gas into the reaction chamber 101. The bacterial filter includes a filter medium and a plastically formed bottom cover and top cover. The bottom cover and top cover are fastened together to form a cavity structure. The filter medium is disposed within the cavity structure. The bottom cover and top cover are respectively connected to an air outlet and an air inlet that communicate with the cavity structure. The filter medium is a filter element or filter membrane used to filter microorganisms. The gas injection assembly includes a disposable syringe or a miniature air pump. The air inlet pipe 8 can be sealed and connected to the air outlet of the bacterial filter, and the nipple of the disposable syringe or the air tube of the miniature air pump can be sealed and connected to the air inlet of the bacterial filter.

[0047] Bacterial filters are standard products available on the market, manufactured by companies such as Yangzhou Qiangjian and Ying Shi Medical; therefore, they will not be discussed further. Disposable syringes and miniature air pumps are also standard products, and will not be discussed further either. Furthermore, the accompanying drawings in the instruction manual do not show views of the fluorometer, bacterial filter, disposable syringe, and miniature air pump. The inventor believes that these are all standard products, and those skilled in the art can understand the specific structure and function of these devices based on actual products.

[0048] The filter element or membrane of the bacterial filter can filter out microorganisms in the air, preventing microorganisms from entering the reaction chamber 101 and affecting the detection process and the accuracy of microbial detection.

[0049] The method of using the rapid microbial detection device in this embodiment is as follows: Open the cover of the fluorometer, place the rapid microbial detection reaction container from Example 1 into the detection chamber on the device body, so that the light surface 102 of the rapid microbial detection reaction container faces the detector in the detection chamber, close the cover, and read the relative fluorescence intensity value (RLU) by the fluorometer.

[0050] After the test is completed, software is used for data processing and result interpretation. The result interpretation criteria are as follows:

[0051] The mean fluorescence signal (RLU) obtained by detecting the fluorescence in MTAT (modified tryptone lecithin Tween broth) or other equivalent media without sample solution is used as the blank fluorescence value of the media. The positive threshold is usually calculated using the following formula:

[0052] Positive threshold = 3 × blank fluorescence value in culture medium

[0053] a) A sample with an RLU value ≥ the positive threshold is considered positive, indicating that microorganisms were detected in the sample under the test conditions.

[0054] b) If the RLU value of the sample is less than the positive threshold, it is considered negative, indicating that no microorganisms were detected in the sample under the test conditions.

[0055] Obviously, it is possible to modify and / or add components to the above-mentioned rapid microbial detection reaction vessel and corresponding method without departing from the field and scope of this utility model.

[0056] It is equally clear that, although this invention has described the rapid microbial detection reaction vessel in detail, those skilled in the art will certainly be able to obtain many other equivalent forms of rapid microbial detection reaction vessels and corresponding methods, which have the features described in the claims and are therefore within the scope of protection defined herein.

Claims

1. A rapid microbial detection reaction vessel, characterized in that: The container includes a main body (1), a conical bottom (3), a drain pipe (4), a sealing cap (5), a drug injection pipe (6), a liquid injection stopper (7), and a filter membrane (10). The main body (1) is a cavity structure. The conical bottom (3) is fixed to the bottom end of the main body (1), so that a closed reaction chamber (101) is formed inside the main body (1). The drain pipe (4) is fixed to the bottom end of the conical bottom (3), and the sealing cap (5) is detachably installed at the end of the drain pipe (4) away from the conical bottom (3). At least one of the injection tubes (6) is fixedly installed on the top surface of the container body (1), and the injection stopper (7) is fixedly installed at the end of the injection tube (6) away from the container body (1). The filter membrane (10) is fixedly installed in the reaction chamber (101) at the end near the conical bottom (3).

2. The rapid microbial detection reaction vessel according to claim 1, characterized in that: The rapid microbial detection reaction container also includes a base (2), which is fixedly connected to the bottom of the container body (1) and integrally formed with the container body (1). The base (2) has a cylindrical structure, and the drain pipe (4) and the sealing cap (5) installed on the drain pipe (4) are located in the inner cavity of the base (2).

3. The rapid microbial detection reaction vessel according to claim 1, characterized in that: The rapid microbial detection reaction container also includes an air inlet pipe (8), which is fixedly installed on the top surface of the container body (1), and the sealing cap (5) is detachably installed at the end of the air inlet pipe (8) away from the container body (1).

4. The rapid microbial detection reaction vessel according to claim 1, characterized in that: The sealing cap (5) includes a sealing part (51) and a handle part (52). The sealing part (51) has a columnar structure and is used to be fitted inside the drain pipe (4) or the air inlet pipe (8). The handle part (52) is fixedly installed at one end of the sealing part (51), and the outer diameter of the handle part (52) is larger than the outer diameter of the sealing part (51).

5. The rapid microbial detection reaction vessel according to claim 1, characterized in that: A support ring (9) is fixedly provided at one end of the reaction chamber (101) near the conical bottom (3). The filter membrane (10) is disposed on the top surface of the support ring (9). The filter membrane (10) is fixedly installed on the support ring (9) by a fixing ring (11).

6. The rapid microbial detection reaction vessel according to claim 1, characterized in that: The container body (1) has a quadrangular prism structure, with one set of opposite sides being smooth (102) and the other set of opposite sides being rough (103).

7. The rapid microbial detection reaction vessel according to claim 1, characterized in that: The injection stopper (7) is a butyl rubber stopper; the pore size of the filter membrane (10) is between 0.22μm and 0.45μm.

8. A rapid microbial detection device, characterized in that: The container includes a liquid injection assembly, a fluorescence spectrometer, and a rapid microbial detection reaction vessel as described in any one of claims 1-7; the liquid injection assembly is used to extract samples or reaction reagents and to inject the samples or reaction reagents into the reaction chamber (101) by puncturing the liquid injection stopper (7); the fluorescence spectrometer is used to measure the fluorescence intensity of the solution in the reaction chamber (101) and to quantify the total amount of microorganisms in the sample.

9. The rapid microbial detection device according to claim 8, characterized in that: The rapid microbial detection device also includes an injection assembly, which is sealed to the air inlet pipe (8) to inject sterile gas into the reaction chamber (101); the liquid injection assembly includes a disposable syringe; the injection assembly includes a disposable syringe or a micro air pump, and the air inlet pipe (8) can be sealed to the nipple of the disposable syringe or the air tube of the micro air pump.

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