Pretreatment tube capable of removing inhibitors in excrement sample and realizing solid-liquid separation

Abstract

The utility model provides a pretreatment tube capable of removing inhibitors in a fecal sample and realizing solid-liquid separation, which comprises a first tube body, a tube cover and a second tube body, the first tube body is used for accommodating the fecal sample and a precipitation reagent, the tube cover can be detached and is hermetically connected to a tube opening of the first tube body, the tube bottom of the second tube body is provided with a filter sheet, and the filter sheet is detachably connected to the tube opening of the second tube body. The second pipe body can be coaxially inserted into the first pipe body along the pipe body center line of the first pipe body, and when the second pipe body is inserted into the first pipe body, supernate formed in the first pipe body can enter the containing space of the second pipe body through the filter piece. According to the utility model, a centrifugal machine does not need to be additionally arranged in a matched full-automatic nucleic acid detection instrument, so that the operation steps are greatly simplified, inhibitors in excrement can be efficiently removed, and nucleic acid can be successfully extracted from supernate after reaction; it can also be ensured that the excrement sample does not make contact with the outside in the pretreatment process, and the excrement sample is not polluted in the pretreatment process.

Description

Technical Field

[0001] This invention belongs to the field of biological sample nucleic acid extraction technology, specifically relating to a pretreatment tube that can remove inhibitors from fecal samples and achieve solid-liquid separation. Background Technology

[0002] Polymerase chain reaction (PCR) is a technique for rapid in vitro DNA amplification, widely used in the genetic diagnosis of infectious and genetic diseases. As an enzymatic reaction, PCR is highly sensitive to inhibitors. However, fecal samples often contain various inhibitors, including bile salts, urea, and heparin. Therefore, removing inhibitors from fecal samples during pretreatment is crucial.

[0003] Currently, the mainstream fecal nucleic acid extraction pretreatment tubes on the market employ a magnetic bead-based inhibitor removal method: the sample is first lysed, then the lysate or supernatant is contacted with a protein precipitant or inhibitor remover, and finally the mixture is separated into solid and liquid phases, with nucleic acids separated from the liquid phase using magnetic beads. The main drawbacks of this method are: cumbersome manual operation, the inability to complete sample lysis, inhibitor removal, and solid-liquid separation in the same sample tube, and a high risk of sample contamination.

[0004] In nucleic acid extraction, the most common solid-liquid separation method is centrifugation. Centrifugation requires placing the pretreatment tubes in a high-speed centrifuge, and the centrifugation time is usually as long as five to ten minutes. It is precisely because of the requirements of centrifugation that fecal samples are difficult to truly utilize in fully automated nucleic acid testing systems. Utility Model Content

[0005] Therefore, the technical problem to be solved by this utility model is to provide a pretreatment tube that can remove inhibitors from fecal samples and achieve solid-liquid separation, so as to solve the technical problems of the existing fecal nucleic acid extraction pretreatment tube using the magnetic bead method, which is cumbersome to operate, and the three steps of sample lysis, inhibitor removal and solid-liquid separation cannot be completed in the same sample tube, which poses a great risk of contamination. The centrifugal separation method requires the use of a high-speed centrifuge, which is difficult to apply in a fully automated nucleic acid detection system.

[0006] To address the aforementioned problems, this invention provides a pretreatment tube capable of removing inhibitors from fecal samples and achieving solid-liquid separation. The tube includes a first tube body and a cap. The first tube body is used to hold the fecal sample and a precipitation reagent. The cap is detachable and sealed to the opening of the first tube body. The invention also includes a second tube body. The bottom of the second tube body has a filter sheet. The second tube body can be coaxially inserted into the first tube body along its centerline. When the second tube body is inserted into the first tube body, the supernatant formed in the first tube body can enter the holding space of the second tube body through the filter sheet.

[0007] In some embodiments, a bottom wall plate with a first through hole and a fixing pad with a second through hole are formed on the bottom of the second tube, and the filter sheet is sandwiched between the bottom wall plate and the fixing pad.

[0008] In some embodiments, the sidewall of the second tube has an annular wall protruding outward from the bottom wall plate, and the annular wall has a plurality of protrusions arranged around the center line of the second tube, and the fixing pad is axially limited to the side of each of the protrusions facing the bottom wall plate.

[0009] In some embodiments, an annular groove is formed on the sidewall of the pipe, surrounding the centerline of the second pipe body, and a sealing ring is clamped between the annular groove and the inner wall of the first pipe body.

[0010] In some embodiments, when the second tube body and the first tube body are in an assembled state, the positions of each of the first through holes and each of the second through holes correspond one-to-one and are connected.

[0011] In some embodiments, the opening of the second tube has an outwardly extending radially outwardly convex ring, the outer diameter of which is greater than the inner diameter of the opening of the first tube but not greater than the outer diameter of the opening of the first tube.

[0012] In some embodiments, the bottom wall of the first tube is an inverted cone shape, wider at the top and narrower at the bottom.

[0013] In some embodiments, the bottom wall of the first tube has a plurality of positioning notches, each of the positioning notches being spaced apart around the center line of the first tube; and / or, the outer surface of the side wall of the first tube is provided with two snap fasteners symmetrical about the center line of the first tube.

[0014] In some embodiments, the cap is threadedly connected to the outer wall of the opening of the first tube body.

[0015] In some embodiments, the ratio of the height to the inner diameter of the first tube is 2 to 7, and the volume is 40 mL to 70 mL; and / or, the ratio of the height to the inner diameter of the second tube is 2 to 7, and the volume is 15 mL to 50 mL.

[0016] This invention provides a pretreatment tube that can remove inhibitors from fecal samples and achieve solid-liquid separation. After the reaction between the lysed sample (i.e., the fecal sample after lysis) and the precipitated inhibitor (i.e., the precipitation reagent stop solution) has been fully completed, a second tube is inserted into the first tube and then filtered through a filter to effectively achieve solid-liquid separation of the mixture. This eliminates the need for an additional centrifuge in the matching fully automated nucleic acid detection instrument, making it highly compatible with existing PCR equipment and greatly simplifying the operation steps. It can efficiently remove inhibitors from feces and successfully extract nucleic acids from the supernatant after the reaction. Using this pretreatment tube also ensures that the fecal sample does not come into contact with the outside environment during pretreatment, preventing contamination and ensuring the success of subsequent nucleic acid extraction and amplification detection. Attached Figure Description

[0017] Figure 1 This is a three-dimensional structural diagram (appearance) of a pretreatment tube that can remove inhibitors from fecal samples and achieve solid-liquid separation according to an embodiment of the present invention;

[0018] Figure 2 for Figure 1 Cross-sectional view of AA in the middle;

[0019] Figure 3 for Figure 1 An exploded view of the processing tube in the diagram.

[0020] The reference numerals in the attached figures are as follows:

[0021] 1. First tube body; 11. Positioning notch; 12. Buckle; 2. Second tube body; 21. Bottom wall plate; 22. Fixing pad; 23. Protrusion; 24. Annular groove; 25. Outwardly convex ring; 3. Tube cap. Detailed Implementation

[0022] See also Figures 1 to 3As shown in the figure, according to an embodiment of the present invention, a pretreatment tube is provided that can remove inhibitors from fecal samples and achieve solid-liquid separation. The tube includes a first tube body 1 and a tube cap 3. The first tube body 1 is used to contain fecal samples and precipitation reagents. The tube cap 3 is detachable and sealed to the opening of the first tube body 1. The pretreatment tube also includes a second tube body 2. The bottom of the second tube body 2 has a filter (not shown in the figure, not labeled). The second tube body 2 can be coaxially inserted into the first tube body 1 along the center line of the first tube body 1. When the second tube body 2 is inserted into the first tube body 1, the supernatant formed in the first tube body 1 (which is formed after adding the precipitation reagent stop solution to the second tube body 2) can enter the containing space of the second tube body 2 through the filter. The filter can be a filter screen, filter membrane, or filter paper with an appropriate mesh size according to actual needs. In some specific embodiments, the filter can be a food-grade non-woven fabric or a polyethersulfone filter membrane.

[0023] In this technical solution, after the reaction between the lysed sample (i.e., the lysed fecal sample) and the precipitation inhibitor (i.e., the precipitation reagent stop solution) has been fully completed, the second tube 2 is inserted into the first tube 1, and the mixture is effectively separated into solid and liquid components by passing it through a filter. This eliminates the need for an additional centrifuge in the matching fully automated nucleic acid detection instrument, making it highly compatible with existing PCR equipment and greatly simplifying the operation steps. It can efficiently remove inhibitors from feces and successfully extract nucleic acids from the supernatant after the reaction. The pretreatment tube of this invention also ensures that the fecal sample does not come into contact with the outside world during the pretreatment process and is not contaminated, thereby ensuring the success of subsequent nucleic acid extraction and amplification detection.

[0024] In some embodiments, a bottom wall plate 21 with a first through hole (not shown in the figure) and a fixing pad 22 with a second through hole (not shown in the figure) are formed on the bottom of the second tube 2, and the filter is sandwiched between the bottom wall plate 21 and the fixing pad 22.

[0025] In this technical solution, the bottom wall plate 21 and the fixed pad 22 together form a clamping mechanism for the filter element, which can ensure reliable support for the filter element, thereby improving the filtration effect and service life of the filter element.

[0026] In one specific embodiment, the thickness of the bottom wall plate 21 is 0.3mm to 0.7mm, and the distance from the bottom wall plate 21 to the lowest end of the second tube 2 is 5mm to 10mm.

[0027] In some embodiments, when the second tube 2 and the first tube 1 are in an assembled state, the positions of each of the first through holes and each of the second through holes are connected one-to-one, which can ensure the flow area of ​​the filter and further improve the solid-liquid separation effect.

[0028] In one specific embodiment, the aforementioned first and second through holes correspond one-to-one in size and shape. As an example, the first through hole consists of a central circular hole and a plurality of fan-shaped holes surrounding the circular hole. The diameter of the circular hole is 3mm to 5mm, and the fan-shaped holes are symmetrically distributed about the center line of the second tube 2, with each fan-shaped hole distributed within an annular region having an inner diameter of 5mm to 20mm and an outer diameter of 35mm to 50mm. The second through hole also has a corresponding circular hole and fan-shaped holes, which will not be described in detail here.

[0029] The aforementioned first tube 1, second tube 2, and tube cap 3 are all made of corrosion-resistant materials, such as polyurethane, polytetrafluoroethylene, polyetheretherketone, and polyphenylene sulfide.

[0030] In some embodiments, the sidewall of the second tube 2 has an annular wall (not shown in the figure) protruding outward from the bottom wall plate 21. The annular wall has a plurality of protrusions 23 arranged around the centerline of the second tube 2. The fixing plate 22 is axially limited on the side of each of the protrusions 23 facing the bottom wall plate 21. The plurality of protrusions 23 reliably limit the axial movement of the fixing plate 22, thereby ensuring reliable support of the filter element by the fixing plate 22. In one specific embodiment, there are three protrusions 23.

[0031] In some embodiments, an annular groove 24 is formed on the sidewall of the pipe, which surrounds the center line of the second pipe body 2, and a sealing ring (not shown or indexed in the figure) is clamped between the annular groove 24 and the inner wall of the first pipe body 1.

[0032] In this technical solution, by setting an annular groove 24 with a trapezoidal cross section on the side wall of the second tube 2, the sealing ring can be reliably clamped in the gap between the second tube 2 and the first tube 1. During the process of the second tube 2 being inserted into the first tube 1, it can be ensured that the sample liquid can be sealed between the second tube 2 and the first tube 1, and the supernatant can be filtered by the filter to achieve solid-liquid separation, thereby improving the separation effect and efficiency.

[0033] See details Figure 2 and Figure 3 As shown, in some embodiments, the opening of the second tube 2 has an outwardly extending outwardly convex ring 25 along its radial direction, the outer diameter of the outwardly extending outwardly convex ring 25 being larger than the inner diameter of the opening of the first tube 1 but not larger than the outer diameter of the opening of the first tube 1.

[0034] In this technical solution, the axial insertion position of the second tube body 2 is accurately positioned by setting an outwardly convex ring 25 at the opening of the second tube body 2.

[0035] In some embodiments, the bottom wall of the first tube 1 is an inverted cone shape that is wider at the top and narrower at the bottom. In a specific embodiment, the apex angle of the aforementioned inverted cone is 130° to 150°, which can facilitate the aggregation of solid components at the bottom after solid-liquid separation and further improve the solid-liquid separation efficiency.

[0036] In some embodiments, the bottom wall of the first tube body 1 has a plurality of positioning notches 11, each of the positioning notches 11 being spaced apart around the center line of the first tube body 1. The positioning notches 11 are used to fix the first tube body 1 in the fully automated nucleic acid detection cartridge. The outer surface of the side wall of the first tube body 1 is provided with two buckles 12 (specifically irregular buckles) symmetrical about the center line of the first tube body 1 for positioning the first tube body 1.

[0037] In some embodiments, the pipe cap 3 is threadedly connected to the outer wall of the pipe opening of the first pipe body 1, which facilitates convenient assembly between the pipe cap 3 and the first pipe body 1.

[0038] In some embodiments, the ratio of the height to the inner diameter of the first tube 1 is 2 to 7, preferably 2.5 to 5, and the volume is 4.0 mL to 7.0 mL, preferably 4.5 mL to 6.0 mL; and / or, the ratio of the height to the inner diameter of the second tube 2 is 2 to 7, preferably 3 to 5, and the volume is 1.5 mL to 5.0 mL, preferably 2.5 mL to 3.5 mL.

[0039] In one specific embodiment, the aforementioned pipe cap 3 has a diameter of 35mm to 50mm and a height of 10mm to 25mm.

[0040] The technical solution of this utility model will be further described below in conjunction with specific usage methods:

[0041] Method 1:

[0042] Add approximately 5 mL of precipitation reagent preservation solution and approximately 1 gram of fecal sample to the first tube 1, then rotate and fix the tube cap 3 onto the first tube 1. Next, use a vortex mixer to vortex the solid-liquid mixture in the pretreatment tube until a homogenate is formed. Then, heat the pretreatment tube at 70°C for approximately 5 minutes using a constant temperature heating instrument. Then, place the pretreatment tube containing the sample and reagents into a fully automated nucleic acid detection instrument. The instrument's robotic arm then rotates to remove the tube cap 3 and inserts the second tube 2 into the first tube 1, slowly lowering the second tube 2 until its opening contacts the opening of the first tube 1. After the above process, the supernatant obtained in the second tube 2 can be automatically processed in the fully automated nucleic acid detection instrument to terminate the subsequent precipitation reaction, extract nucleic acid, and perform amplification detection.

[0043] Method 2:

[0044] This procedure can be performed manually without the use of fully automated nucleic acid testing instruments. The specific implementation is as follows: Add approximately 5 mL of precipitation reagent preservation solution to the first tube 1, then add approximately 1 gram of fecal sample and an appropriate amount of distilled water. Rotate and fix the tube cap 3 onto the first tube 1. Then, use a vortex mixer to vortex and homogenize the solid-liquid mixture in the pretreatment tube until a homogenate is formed. Next, heat the pretreatment tube at 70°C for approximately 5 minutes using a constant temperature heating instrument. Then, rotate and remove the tube cap 3, and insert the second tube 2 into the first tube 1, slowly lowering the second tube 2 until its opening contacts the opening of the first tube 1. Then, add approximately 5 mL of precipitation reagent stop solution to the second tube 2 to terminate the precipitation and lysis reactions in the supernatant. After the above process, the supernatant obtained in the second tube 2 can be used for subsequent nucleic acid extraction and amplification detection.

[0045] The working principle of this invention is explained as follows: In the first tube 1, the bacteria in the sample are fully lysed by the lysis reagent, exposing their genetic material to the system; simultaneously, the PCR inhibitors in the fecal sample are fully precipitated by the flocculation reagent. After the second tube 2 slowly descends, because the sealing ring can effectively seal the annular gap between the first tube 1 and the second tube 2, as the pressure inside the first tube 1 gradually increases, the reacted system can gradually pass through the fixing plate 22, the filter sheet, and the bottom wall plate 21 of the second tube 2 into the second tube 2, filtering out all precipitates and colloidal particles. A supernatant is obtained in the second tube 2, which contains relatively pure nucleic acid extract, and can then be used for subsequent nucleic acid extraction and amplification detection.

[0046] It will be readily understood by those skilled in the art that the aforementioned advantageous methods can be freely combined and superimposed without conflict.

[0047] The above are merely preferred embodiments of this utility model and are not intended to limit the scope of this utility model. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this utility model should be included within the protection scope of this utility model. The above are only preferred embodiments of this utility model. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this utility model, and these improvements and modifications should also be considered within the protection scope of this utility model.

Claims

1. A pre-treatment tube that can remove inhibitors in a fecal sample and achieve solid-liquid separation, characterized by, The device includes a first tube (1) and a cap (3). The first tube (1) is used to hold a fecal sample and a precipitation reagent. The cap (3) is detachable and sealed to the opening of the first tube (1). The device also includes a second tube (2). The bottom of the second tube (2) has a filter. The second tube (2) can be coaxially inserted into the first tube (1) along the center line of the first tube (1). When the second tube (2) is inserted into the first tube (1), the supernatant formed in the first tube (1) can enter the holding space of the second tube (2) through the filter.

2. The pre-treatment tube of claim 1, wherein, The bottom of the second tube (2) is formed with a bottom wall plate (21) having a first through hole and a fixing pad (22) having a second through hole connected to the outside of the bottom wall plate (21), and the filter is held between the bottom wall plate (21) and the fixing pad (22).

3. The pre-treatment tube of claim 2, wherein, The second tube (2) has a sidewall with an annular wall protruding from the outside of the bottom wall plate (21). The annular wall has a plurality of protrusions (23) arranged around the center line of the second tube (2). The fixing pad (22) is axially limited to the side of each of the protrusions (23) facing the bottom wall plate (21).

4. The pre-treatment tube of claim 3, wherein, An annular groove (24) is formed on the side wall of the pipe, which surrounds the center line of the second pipe body (2). A sealing ring is clamped between the annular groove (24) and the inner wall of the first pipe body (1).

5. The pre-treatment tube of claim 2, wherein, When the second tube (2) and the first tube (1) are in the assembled state, the positions of each first through hole and each second through hole are connected in a one-to-one correspondence.

6. The pre-treatment tube of claim 1, wherein, The second tube (2) has an outwardly convex ring (25) extending radially outward, the outer diameter of which is greater than the inner diameter of the first tube (1) and not greater than the outer diameter of the first tube (1).

7. The pre-treatment tube of claim 1, wherein The bottom wall of the first tube (1) is an inverted cone shape that is larger at the top and smaller at the bottom.

8. The pre-treatment tube of claim 1, wherein, The bottom wall of the first tube (1) has a plurality of positioning notches (11), and each positioning notch (11) is spaced apart around the center line of the first tube (1); and / or, the outer surface of the side wall of the first tube (1) is provided with two buckles (12) symmetrical about the center line of the first tube (1).

9. The pre-treatment tube of claim 1, wherein, The pipe cap (3) is threadedly connected to the outer wall of the opening of the first pipe body (1).

10. The pre-treatment tube of claim 1, wherein, The height-to-inner-diameter ratio of the first tube (1) is 2 to 7, and the volume is 4.0 mL to 7.0 mL; and / or the height-to-inner-diameter ratio of the second tube (2) is 2 to 7, and the volume is 1.5 mL to 50 mL.

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