Centrifugal tube

Abstract

The utility model discloses a centrifugal tube which is characterized by comprising a centrifugal tube cap (1), a pressing cover (2) matched with the centrifugal tube cap and a centrifugal tube (3), an external thread is arranged on the outer wall of a tube opening of the centrifugal tube (3); internal threads are arranged on the groove wall of one side, close to the outer edge of the centrifugal tube (3), of the centrifugal tube cap (1), a first through hole (11) is formed in the centrifugal tube cap (1), one end of the first through hole (11) is sealed by aluminum foil (4), and the other end of the first through hole (11) is in contact with the pressing cover (2) to form a liquid storage cavity (12); the pressing cover (2) is composed of a pipe (21) and a cover (22) sealing one end of the pipe (21), and the length of the pipe (21) is larger than the depth of one end of the first through hole (11) sealed by the aluminum foil (4). The kit disclosed by the utility model can be matched with an automatic pathogen extraction device, and reagents can be added into a sample after the kit is pressed, so that pollution is avoided.

Description

Technical Field

[0001] The utility model relates to the field of pathogen detection, in particular to a centrifuge tube for extracting pathogens. Background Art

[0002] Infectious diseases are common clinical diseases. In the diagnosis and treatment of infectious diseases, pathogen diagnosis is a key link, and conventional pathogen detection methods involve microbial culture. Microbial culture methods are time-consuming (usually 1-5 days), which leads to untimely results; the positive rate is low (the detection rate is less than 15%), which leads to incomplete results or poor sensitivity, and there is a possibility of misleading diagnosis; the operation process is cumbersome and consumes a lot of manpower. Furthermore, the differences in the various pathogens present in the sample lead to differences in culture conditions, and the composition of the dominant pathogens after culture may not reflect the original state in the sample. Therefore, the microbial culture method is difficult to meet clinical needs.

[0003] For example, in the case of blood infections, the pathogen content in the sample is low (1-10 CFU / mL), and the long culture process results in a time interval of 1-5 days between sampling and providing results. Shortening the diagnosis time of infectious diseases can greatly improve patient survival rates. In addition, before a reliable diagnosis is obtained, the usual treatment involves giving the patient a broad-spectrum non-targeted antibiotic. However, since various microorganisms can develop multidrug resistance, there is a risk of other serious consequences even if the disease is alleviated.

[0004] Therefore, rapid early diagnosis helps to improve the survival rate of infected patients and reduce the disability rate of survivors. Clinically, there is an urgent need for methods and corresponding products that can provide reliable diagnostic information in a shorter time, such as a few hours, with high sensitivity and small sample requirements, such as centrifuge tubes that are compatible with automated pathogen extraction devices. Utility Model Content

[0005] In order to improve the above technical problems, on the one hand, the utility model provides a centrifuge tube, characterized in that:

[0006] It comprises a centrifuge tube cap (1), a press cover (2) matched with the centrifuge tube cap, and a centrifuge tube body (3);

[0007] The outer wall of the tube mouth of the centrifugal tube body (3) is provided with an external thread;

[0008] The centrifuge tube cap (1) is provided with an internal thread on a groove wall on one side close to the outer edge of the centrifuge tube body (3), and a first through hole (11) is provided inside. One end of the first through hole (11) is sealed by an aluminum foil (4), and the other end contacts the press cover (3) to form a liquid storage cavity (12);

[0009] The push cover (2) is composed of a tube (21) and a cover (22), wherein the cover (22) seals one end of the tube (21), wherein the length of the tube (21) is greater than the depth of one end of the first through hole (11) sealed by the aluminum foil (4).

[0010] In one embodiment, the liquid storage chamber (12) in the aforementioned centrifuge tube contains acid-washed glass beads, and after the cover (2) is pressed to pierce the aluminum foil (4), the acid-washed glass beads contained in the liquid storage chamber (12) enter the centrifuge tube.

[0011] In one embodiment, the cross section of the capped tube (21) of the aforementioned centrifuge tube near the aluminum foil (4) is elliptical.

[0012] In one embodiment, the centrifuge tube cap (1), the press cover (2) matched with the centrifuge tube cap, and the centrifuge tube body (3) in the centrifuge tube are all made of polypropylene material.

[0013] Beneficial effects

[0014] The centrifuge tube disclosed in the utility model can be matched with an automated pathogen extraction device, facilitating automated operation and ensuring the smoothness of mechanized operation; reaction components can be added to the sample after the centrifuge tube is pressed, which can avoid contamination caused by opening and closing the lid; glass beads are scattered particles that are easy to scatter everywhere, and accommodating them in the centrifuge tube in advance can save sample addition time. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The invention is a schematic diagram of the overall structure of a centrifuge tube cap (1), a push cover (2) matched with the centrifuge tube cap, and a centrifuge tube body (3).

[0016] Figure 2 It is a side cross-sectional view of a centrifuge tube cap (1), a push cover (2) matched with the centrifuge tube cap, and a centrifuge tube body (3).

[0017] Figure 3 It is a schematic diagram of a cover (2), which includes a tube (21) and a cover (22) that seals one end of the tube (21).

[0018] Figure 4 The test results of Test Example 1 on Escherichia coli are shown.

[0019] Figure 5 The test results of Test Example 1 on Klebsiella pneumoniae are shown.

[0020] Figure 6 The test results of Test Example 1 on Staphylococcus aureus are shown.

[0021] Figure 7 The test results of Test Example 1 on Candida albicans are shown. DETAILED DESCRIPTION

[0022] The following will further describe the technical solutions of the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are merely illustrative and explain the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included within the scope of protection intended by the present invention.

[0023] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0024] definition

[0025] As used herein, a "sample tube" is defined as a tube containing a sample.

[0026] Sample preparation

[0027] First, count Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, or Candida albicans, and then dilute with sterile water to 100,000 (E5) cells / μL. Then, dilute 10-fold to 10,000 (E4) cells / μL, 1,000 (E3) cells / μL, 100 (E2) cells / μL, and 10 (E1) cells / μL.

[0028] Bloodstream infection sample separation, enrichment, extraction and detection experimental group and control group settings:

[0029] Experimental group: Three sample tubes, each containing 6 mL of pathogen-free negative blood, were divided into three groups, one for each. 6 μL of 1000 (E3) cells / μL, 100 (E2) cells / μL, and 10 (E1) cells / μL Escherichia coli (or Klebsiella pneumoniae / Staphylococcus aureus / Candida albicans) were added to each group, respectively. After mixing, they were used as simulated positive samples for later use. At the same time, one sample tube containing 6 mL of pathogen-free negative blood was prepared as a negative extraction control group (BNTC).

[0030] Control group: Six centrifuge tubes containing 250 μL of pathogen lysis solution and 50 μL of microbial sedimentation aid were divided into three groups of two. 3 μL of 1000 (E3) cells / μL, 100 (E2) cells / μL, and 10 (E1) cells / μL of Escherichia coli (or Klebsiella pneumoniae / Staphylococcus aureus / Candida albicans) were added to each group, respectively. The mixture was used as the control sample for later use.

[0031] Treatment of bloodstream infection control group:

[0032] To the control group sample, 25 mg φ0.1 mm and 25 mg φ1 mm acid-washed glass beads were added, vortexed for 5 minutes, heated at 100°C for 5 minutes, cooled at room temperature for 3 minutes, and centrifuged at 14,000 × g for 2 minutes. The supernatant was collected to obtain the nucleic acid of the control group sample.

[0033] Example 1: Automated pathogen separation and extraction process

[0034] 1. Prepare the corresponding treatment solution according to the following composition:

[0035] Microbial sedimentation aid: Novec7500.

[0036] The first selective lysis solution is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 8), 0.03% sodium chloride, 0.035% ammonium sulfate, 0.02% sodium citrate, and 0.025% sorbitol.

[0037] The second selective lysis solution is an aqueous solution containing the following components: 5 mM Tris hydrochloric acid buffer (pH 7), 0.03% potassium chloride, 0.035% magnesium sulfate, 0.02% glucose, and 0.025% betaine.

[0038] The third selective lysis solution is an aqueous solution containing the following components: 5 mM potassium phosphate buffer (pH 8), 0.03% potassium bicarbonate, 0.035% magnesium chloride, 0.02% mannitol, and 0.025% BSA.

[0039] The washing solution is an aqueous solution containing the following components: 0.1% phosphate buffer (pH 7.5), 0.9% sodium chloride, 3% ammonium sulfate, and 0.1% trisodium citrate.

[0040] Pathogen lysis solution: 0.2% Tris-HCl buffer (pH 9), 0.15% potassium chloride, 3% Triton X-100 and 0.05% polypropylene glycol.

[0041] A microbial sedimentation aid is also added to the microbial sedimentation aid reservoir, and the first, second, and third selective lysis solutions, a wash solution, and a pathogen lysis solution are respectively added to the first, second, and third treatment solution reservoirs, a wash solution reservoir, and a pathogen lysis solution reservoir. The first, second, and third treatment solution reservoirs, a wash solution reservoir, and a pathogen lysis solution reservoir are located on the same orifice plate. After the aforementioned liquids are added, the orifice plate is placed in a suitable position.

[0042] 2. Place approximately 6 mL of the collected sample into a sample tube, cover the sample tube, prepare two empty 5 mL centrifuge tubes, and cover the centrifuge tubes.

[0043] 3. Turn on the control unit and automatically execute the following procedures:

[0044] (1) Use the cover opening and closing unit to place the sample tube in a fixed position, then use the identification unit to scan the identification code on the sample tube, then use the cover opening and closing unit to place the sample tube in the shaking unit, and shake the sample tube upside down. Start the cover opening and closing unit to open the cover of the sample tube, use the liquid supply / suction unit to suck out about 3 ml of sample from the sample tube and supply it to the centrifuge tube through the first liquid adding gun, then use the positioning mechanism to position the liquid supply / suction unit to the first treatment liquid storage tank and supply the first selective lysis liquid to the centrifuge tube through the first liquid adding gun, then use the positioning mechanism to position the liquid supply / suction unit to the microbial precipitation agent storage tank and supply the microbial precipitation agent to the sample tube through the second liquid adding gun, start the cover opening and closing unit again to close the cover of the centrifuge tube, move the centrifuge tube to the oscillation unit for vortex oscillation, then move the centrifuge tube to the centrifugation unit, start the centrifuge for the first centrifugation, after the first centrifugation is completed, start the cover opening and closing unit to open the cover of the centrifuge tube, use the first liquid extraction gun to extract the supernatant in the centrifuge tube and discard it into the waste liquid tank;

[0045] (2) positioning the liquid supply / suction unit to the second treatment liquid storage tank through the positioning mechanism and supplying the second selective lysis liquid into the centrifuge tube through the first liquid adding gun, starting the cover opening and closing unit again to close the cover of the centrifuge tube, moving the centrifuge tube to the oscillation unit for vortex oscillation, then moving the centrifuge tube to the centrifugation unit, starting the centrifuge for a second centrifugation, and after the second centrifugation is completed, starting the cover opening and closing unit to open the cover of the centrifuge tube, using the first liquid taking gun to suck the supernatant in the centrifuge tube and discarding it into the waste liquid tank;

[0046] (3) positioning the liquid supply / suction unit to the third treatment liquid storage tank through the positioning mechanism and supplying the third selective lysis liquid into the centrifuge tube through the first liquid adding gun, starting the cover opening and closing unit again to close the cover of the centrifuge tube, moving the centrifuge tube to the oscillation unit for vortex oscillation, then moving the centrifuge tube to the centrifugation unit, starting the centrifuge for the third centrifugation, and after the third centrifugation is completed, starting the cover opening and closing unit to open the cover of the centrifuge tube, using the first liquid taking gun to suck the supernatant in the centrifuge tube and discarding it into the waste liquid tank;

[0047] (4) positioning the liquid supply / suction unit to the washing liquid storage tank through the positioning mechanism, and supplying the washing liquid to the centrifuge tube through the first liquid adding gun, starting the cover opening and closing unit again to close the cover of the centrifuge tube, moving the centrifuge tube to the oscillation unit for vortex oscillation, and then moving the centrifuge tube to the centrifugation unit, starting the centrifuge for the fourth centrifugation, and after the fourth centrifugation is completed, starting the cover opening and closing unit to open the cover of the centrifuge tube, using the first liquid extraction gun to suck the supernatant in the centrifuge tube and discarding it into the waste liquid tank;

[0048] (5) positioning the liquid supply / suction unit to the pathogen lysate storage tank through the positioning mechanism, and supplying the pathogen lysate to the centrifuge tube through the first liquid adding gun, starting the cover opening and closing unit again to close the cover of the centrifuge tube, moving the centrifuge tube to the oscillation unit for vortex oscillation, and then moving the centrifuge tube to the centrifugation unit, starting the centrifuge for the fifth centrifugation, and after the fifth centrifugation is completed, starting the cover opening and closing unit to open the cover of the sample tube, using the first liquid extraction gun to suck the supernatant in the centrifuge tube and discarding it into the waste liquid tank;

[0049] (6) Use the opening and closing cover unit to close the cover of the centrifuge tube, then place the centrifuge tube into the heating unit, add acid-washed glass beads, shake, heat, and centrifuge the centrifuge tube to obtain a pathogen analysis sample.

[0050] The specific extraction steps are shown in Table 1 below (bloodstream infection samples).

[0051]

[0052] Test Example 1: Bloodstream infection samples obtained in Example 1

[0053] Probe-based fluorescence quantitative PCR detection was performed, and a negative amplification control (NTC) was performed at the same time. The system and procedure were the same as those in Test Example 1.

[0054] See also Figures 4 to 7 Among them, C-E3, C-E2, and C-E1 are control groups, which are the corresponding amplification results after direct extraction of nucleic acid from Escherichia coli (Klebsiella pneumoniae / Staphylococcus aureus / Candida albicans) with initial concentrations of 1000 / μL, 100 / μL, and 10 / μL, respectively; E3, E2, and E1 are experimental groups, which are the amplification results of nucleic acid extracted from Escherichia coli (Klebsiella pneumoniae / Staphylococcus aureus / Candida albicans) with initial concentrations of 1000 / μL, 100 / μL, and 10 / μL, respectively; BNTC: is the amplification result of nucleic acid extracted after separation and enrichment of sterile negative blood; NTC: is the negative amplification control.

[0055] Results: Neither NTC nor BNTC amplified, demonstrating effective amplification. The isolation results for the four bacterial strains in the experimental group were nearly identical to those in the control group, generally conforming to the principle of fluorescence amplification. Overall, the device and method described in Example 1 can detect at least 10 pathogens in bloodstream infection samples, meeting the requirements for direct bloodstream infection detection.

[0056] The above describes the implementation methods of the present invention. However, the present invention is not limited to the above implementation methods. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A centrifuge tube, characterized in that: It comprises a centrifuge tube cap (1), a press cover (2) matched with the centrifuge tube cap, and a centrifuge tube body (3); The outer wall of the tube mouth of the centrifugal tube body (3) is provided with an external thread; The centrifuge tube cap (1) is provided with an internal thread on a groove wall on one side close to the outer edge of the centrifuge tube body (3), and a first through hole (11) is provided inside. One end of the first through hole (11) is sealed by an aluminum foil (4), and the other end contacts the press cover (2) to form a liquid storage cavity (12); The push cover (2) is composed of a tube (21) and a cover (22), wherein the cover (22) seals one end of the tube (21), and the length of the tube (21) is greater than the depth of one end of the first through hole (11) sealed by the aluminum foil (4).

2. The centrifuge tube according to claim 1, wherein: The liquid storage chamber (12) contains acid-washed glass beads. After the cover (2) is pressed to pierce the aluminum foil (4), the acid-washed glass beads contained in the liquid storage chamber (12) enter the centrifuge tube.

3. The centrifuge tube according to claim 1, wherein: The cross section of the tube (21) close to the aluminum foil (4) is elliptical.

4. A centrifuge tube according to claim 1, characterized in that: The centrifuge tube cap (1), the push cover (2) matched with the centrifuge tube cap, and the centrifuge tube body (3) are all made of polycarbonate material.

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