Kit for extracting pathogens

Abstract

The utility model discloses a kit for extracting pathogens, which comprises a square box body 1 and an eight-joint reagent containing tube 2, eight reagent containing pools 3 are arranged in the eight-joint reagent containing tube 2 at intervals, each side surface of the box body 1 is provided with eight convex edges 11 to form four grooves 12, and a partition plate 13 is arranged between every two adjacent grooves. The groove 12 is used for accommodating a plate 21 on the eight-joint reagent accommodating tube 2; the eight reagent containing pools 3 comprise seven large pools and a small pool, and a first selective lysis solution, a second selective lysis solution, a third selective lysis solution and a washing solution are sequentially contained in the seven large pools from the direction close to the small pool; and a microbial settling agent is accommodated in the small pool. The kit disclosed by the utility model can be matched with an automatic pathogen extraction device, and pathogens in a sample can be conveniently and automatically extracted, so that labor is saved, and pollution caused by manual operation is avoided.

Description

Technical Field

[0001] The utility model relates to the field of pathogen detection, in particular to a kit 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 bloodstream infections, the pathogen content in the sample is low (1-10 CFU / mL), and the lengthy culture process results in a typical time interval of 1-5 days between sampling and providing results. In contrast, studies have shown that patients with severe bloodstream infections or shock-prone bloodstream infections have a 7.6% increased likelihood of death per hour without prompt antibiotic treatment. Furthermore, if antibiotics are not used within the first 6 hours of identifying hypotension, patient survival rates are significantly reduced. Therefore, shortening the time it takes to diagnose infectious diseases can significantly improve patient survival rates. Furthermore, before a reliable diagnosis is achieved, typical treatment involves administering broad-spectrum, non-targeted antibiotics to the patient. However, because various microorganisms can develop multidrug resistance, even if the disease is alleviated, there is a risk of other serious consequences.

[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 test kits that are compatible with automated pathogen extraction devices. Utility Model Content

[0005] In order to improve the above technical problems, the utility model provides a kit for extracting pathogens, characterized in that: it includes a square box body 1 and an eight-way reagent holding tube 2, eight reagent holding pools 3 are provided in the eight-way reagent holding tube 2, and eight convex ridges 11 are provided on each of the two side surfaces of the box body 1, forming four grooves 12, and partitions 13 are provided between adjacent grooves, and the grooves 12 are used to accommodate the plates 21 on the eight-way reagent holding tube 2;

[0006] The eight reagent holding pools 3 include seven large pools and one small pool. The seven large pools contain the first selective lysis solution, the second selective lysis solution, the third selective lysis solution, the washing solution and the pathogen lysis solution in order from the direction close to the small pool; the small pool contains a microbial precipitation aid.

[0007] In one embodiment of the present invention, there are two types of washing liquids, namely a first washing liquid and a second washing liquid. The seven large pools of the kit contain a first selective lysis liquid, a second selective lysis liquid, a third selective lysis liquid, a first washing liquid, a second washing liquid and a pathogen lysis liquid in order from the direction close to the small pool; the small pool contains a microbial precipitation aid.

[0008] In one embodiment of the present invention, the box body 1 and the eight-way reagent containing tube 2 of the reagent kit are made of plastic.

[0009] In one embodiment of the present invention, the eight-reagent containing tube 2 of the reagent kit is covered with aluminum foil.

[0010] Beneficial effects

[0011] The kit disclosed in the utility model can be matched with an automated pathogen extraction device to facilitate the automated extraction of pathogens from samples, thereby saving labor and avoiding contamination caused by manual operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic diagram of the overall structure of the utility model.

[0013] Figure 2 The test results of Test Example 1 on Escherichia coli are shown.

[0014] Figure 3 The test results of Test Example 1 on Klebsiella pneumoniae are shown.

[0015] Figure 4 The test results of Test Example 1 on Staphylococcus aureus are shown.

[0016] Figure 5 The test results of Test Example 1 on Candida albicans are shown. DETAILED DESCRIPTION

[0017] 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.

[0018] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0019] Sample preparation

[0020] 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.

[0021] Bloodstream infection sample separation, enrichment, extraction and detection experimental group and control group settings:

[0022] Experimental group: Each sample tube was filled with 6 mL of pathogen-free negative blood. The six sample tubes were divided into three groups, one for each group. 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, a sample tube containing 6 mL of pathogen-free negative blood was prepared as a negative extraction control group (BNTC).

[0023] 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.

[0024] Treatment of bloodstream infection control group:

[0025] 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.

[0026] Example 1: Automated pathogen separation and extraction process

[0027] 1. Prepare the corresponding treatment solution according to the following composition:

[0028] Microbial sedimentation aid: Novec7500.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Pathogen lysis solution: 0.2% Tris-HCl buffer (pH 9), 0.15% potassium chloride, 3% Triton X-100 and 0.05% polypropylene glycol.

[0034] Separately, a microbial sedimentation aid is 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 added to the first, second, and third treatment solution reservoirs, respectively. The microbial sedimentation aid reservoir, the first, second, and third treatment solution reservoirs, the wash solution reservoir, and the pathogen lysis solution reservoir are located on the same orifice plate. After the aforementioned liquids are added, the orifice plate is positioned appropriately.

[0035] 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.

[0036] 3. Turn on the control unit and automatically execute the following procedures:

[0037] (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;

[0038] (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;

[0039] (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;

[0040] (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;

[0041] (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;

[0042] (6) Use the lid opening and closing unit to close the lid 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.

[0043] The specific extraction steps are shown in Table 1 below (bloodstream infection samples).

[0044] Therefore, the control unit sends instructions to execute the above procedures (1), (2), (3), (3), (4), (4), (5) and (6) in sequence to obtain an extract, and the supernatant of the extract is transferred to a clean centrifuge tube and temporarily stored at 4°C or directly used in the test case.

[0045]

[0046] Test Example 1: Bloodstream infection samples obtained in Example 1

[0047] 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.

[0048] See also Figures 2 to 5 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.

[0049] 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.

[0050] 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 kit for extracting pathogens, characterized in that: The invention comprises a square box body (1) and an eight-joint reagent holding tube (2), wherein eight reagent holding pools (3) are arranged at intervals in the eight-joint reagent holding tube (2), and eight convex ridges (11) are provided on each of the two side surfaces of the box body (1), forming four grooves (12), and partitions (13) are provided between adjacent grooves, and the grooves (12) are used to accommodate the plates (21) on the eight-joint reagent holding tube (2); The eight reagent holding pools (3) include seven large pools and one small pool. The seven large pools sequentially contain a first selective lysis solution, a second selective lysis solution, a third selective lysis solution and a washing solution from the direction close to the small pool; and the small pool contains a microbial precipitation aid.

2. The kit for extracting pathogens according to claim 1, characterized in that: The box body (1) and the eight-link reagent containing tube (2) are made of plastic.

3. The kit for extracting pathogens according to claim 1 or 2, characterized in that: The eight-joint reagent containing tube (2) is covered with aluminum foil.

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