Integrated sample collection and extraction-free nucleic acid release device

Abstract

The utility model discloses an integrated sample collection and extraction-free nucleic acid release device which comprises an injector needle tube with a liquid injection port at the front end, and a piston push rod adaptively inserted into the injector needle tube, the interior of a tube cavity of the syringe needle tube is divided into a front cavity and a rear cavity through a partition plate, a first filter is arranged on the partition plate, and a first cover plate is hinged to the partition plate located on the side of the front cavity; a sample adding port communicated with the rear chamber is formed in the tube wall of the injector needle tube, a sample reaction tube with a horn mouth structure is arranged on the sample adding port in an outward extending manner, a sealing cover is adaptively buckled at the outer end of the sample reaction tube, a second filter is arranged in the sample adding port, and a second cover plate is hinged to the sample adding port positioned on the rear chamber side; the hinge point of the second cover plate is located on the side, away from the partition plate, of the sample adding opening. The kit has the advantages that the kit is convenient to use, low in cost and high in compatibility, the pathogen sensitivity and specificity of a PCR amplification experiment are improved, and the pathogen nucleic acid diagnosis accuracy is improved.

Description

Technical Field

[0001] This utility model relates to the field of molecular biology testing technology, and in particular to an integrated sample collection and extraction-free nucleic acid release device. Background Technology

[0002] Molecular diagnostics is a diagnostic technique that detects changes in the structure or expression levels of genetic material within an organism to predict and diagnose diseases. PCR detection is a crucial technique in molecular diagnostics, and PCR amplification is a simplified method within PCR that allows for direct detection without nucleic acid separation and extraction. Currently, extraction-free amplification systems still contain a significant amount of inhibitors during sample processing and nucleic acid release, which considerably impacts the sensitivity and accuracy of the amplification system. Therefore, effectively removing these inhibitors from the sample itself is a key issue that current PCR amplification technologies need to address.

[0003] There are few existing nucleic acid sampling and release devices specifically designed for PCR amplification technology. Most of them involve adding a release agent to simpler samples, such as throat swabs, to achieve nucleic acid release. For example, the "Nucleic Acid Release Sampling Device" with patent number CN202121729338.2 is mainly used for sampling after nucleic acid release. The device contains a nucleic acid release agent, which releases the nucleic acid upon contact with the sample. While this device is convenient for sampling, it does not include the sampling process itself. Furthermore, interfering substances remain in the liquid after nucleic acid release, and the liquid composition is relatively complex, which can significantly affect the sensitivity and specificity of nucleic acid detection. For example, the patent CN202222117307.2, "A Nucleic Acid Extraction Tube," uses a release agent and an adsorbent added inside the tube to perform crude extraction and detection of nucleic acids. This extraction tube does not contain a sampling module and cannot collect samples; it can only process samples by adding them through a transfer tube. In addition, after the nucleic acid is released, only some impurities in the liquid are adsorbed, leaving many impurities of different particle sizes, which still cause significant interference to the amplification test results. Furthermore, after the nucleic acid is released, the crude nucleic acid liquid needs to be manually aspirated again using a pipette or other device, making the operation cumbersome. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing an integrated sample collection and extraction-free nucleic acid release device.

[0005] To achieve the above objectives, the present invention can adopt the following technical solution:

[0006] The integrated sample collection and extraction-free nucleic acid release device of this utility model includes a syringe needle with an injection port at the front end, and a piston rod adapted to be inserted into the syringe needle. The syringe needle is divided into a front chamber and a rear chamber by a partition plate. A first filter for connecting the front chamber and the rear chamber is provided on the partition plate. A first cover plate for sealing the first filter is hinged to the partition plate on the side of the front chamber. A sample dispensing port communicating with the rear chamber is opened on the tube wall of the syringe needle. A sample reaction tube with a flared mouth structure is provided outward from the sample dispensing port. A sealing cap is adapted to be fastened to the outer end of the sample reaction tube. A second filter is provided in the sample dispensing port. A second cover plate for sealing the second filter is hinged to the sample dispensing port on the side of the rear chamber. The hinge point of the second cover plate is located on the side of the sample dispensing port away from the partition plate.

[0007] Furthermore, for ease of operation, sample processing solution should be pre-encapsulated in the sample reaction tube, and nucleic acid release solution should be pre-encapsulated in the rear chamber. This allows samples requiring nucleic acid sampling to be directly added to the sample reaction tube for pre-treatment with the sample processing solution, and then directly drawn into the rear chamber of the syringe needle to react with the nucleic acid release solution to release nucleic acid. At this time, an annular retainer for securing the piston rod should be fitted onto the syringe needle, and an opening slit for removal from the piston rod should be provided on one side of the annular retainer to prevent accidental squeezing of the piston rod during transfer or transportation, which could cause the nucleic acid release solution to be squeezed out of the syringe needle.

[0008] Furthermore, both the first filter and the second filter can be commercially available disposable needle filters. The first filter has a membrane pore size of 0.45-1 micrometer, which can effectively filter impurities contained in the sample after reaction with the sample processing solution. The second filter has a membrane pore size of 0.22-0.45 micrometers, which can effectively filter interfering substances such as proteins and bacteria after the sample has completed nucleic acid release, reduce inhibitors entering the PCR amplification system, improve the sensitivity and specificity of the amplification system in detecting pathogens, and improve the accuracy of pathogen nucleic acid diagnosis.

[0009] The advantages of this invention are that it can complete sample pretreatment, nucleic acid release, and filtration with a single device. It is convenient to use, low in cost, and highly compatible, applicable to the collection and processing of various samples such as swabs, sputum, and lavage fluid. At the same time, it can effectively lyse infectious disease pathogens in the samples to achieve complete inactivation, reduce inhibitors entering the amplification system, improve the sensitivity and specificity of pathogens in PCR amplification experiments, and enhance the accuracy of pathogen nucleic acid diagnosis. In addition, there is no sample exposure during the entire operation, which greatly improves the biosafety of sample nucleic acid release operations in PCR amplification experiments. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of the structure of this utility model.

[0011] Figure 2 yes Figure 1 Axonometric view of the middle ring-shaped card holder.

[0012] Figure 3 This is the positive amplification pattern for pulmonary tuberculosis detection in Example 1.

[0013] Figure 4 This is the positive amplification pattern for influenza A detection in Example 2. Detailed Implementation

[0014] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present utility model.

[0015] It should be noted that all directional indicators (such as up, down, left, right, front, back, etc.) in this utility model embodiment are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indicator will also change accordingly.

[0016] Furthermore, in this utility model, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature.

[0017] like Figure 1As shown, the integrated sample collection and extraction-free nucleic acid release device of this utility model includes a syringe needle 1, the front end of which has an injection port 2, and a piston rod 3 consisting of a rubber piston head, a straight rod and a push handle is adapted and inserted into the syringe needle 1.

[0018] The syringe needle tube 1 is divided by a partition plate 4 into a front chamber 5 that is in communication with the injection port 2, and a rear chamber 6 in which the piston rod 3 is fitted from back to front. The partition plate 4 is provided with a first filter 7 for connecting the front chamber 5 and the rear chamber 6. The first filter 7 is a disposable needle filter with a filter membrane pore size of 0.22~0.45 micrometers. A first cover plate 8 for sealing the first filter 7 is hinged to the partition plate 4 located on the side of the front chamber 5.

[0019] A sample inlet 9, communicating with the rear chamber 6, is provided on the wall of the syringe needle 1. The sample inlet 9 is located near the partition plate 4, and a sample reaction tube 10 extends outward from it. The sample reaction tube 10 has a flared structure with a gradually increasing diameter. A sealing cap 11 is fitted to the outer end of the sample reaction tube 10 to seal its outer port. A second filter 12 is provided inside the sample inlet 9. The second filter 12 is a disposable needle-type filter with a filter membrane pore size of 0.45~1 micrometer, located on the side of the rear chamber 6. A second cover plate 13 for sealing the second filter 12 is hinged to the sample port 9. The hinge point of the second cover plate 13 is located on the side of the sample port 9 away from the partition plate 4, so that when the piston rod 3 is pushed inward, the rubber piston head on the piston rod 3 can just push the second cover plate 13 to rotate towards the sample port 9 to seal the second filter 12. When the second cover plate 13 seals the second filter 12, its lower surface is just flush with the cavity wall of the rear chamber 6 to prevent the second cover plate 13 from blocking the piston rod 3 from moving back and forth.

[0020] In addition, a certain amount of sample processing solution 14 is pre-packaged in the sample reaction tube 10, and a certain amount of nucleic acid release solution 15 is pre-packaged in the rear chamber 6 located in front of the piston rod 3. Since the rubber piston head at the front end of the piston rod 3 is tightly inserted into the cavity wall of the rear chamber 6, a closed chamber can be formed in front of the rubber piston head at the front end of the piston rod 3 in the rear chamber 6, ensuring that the nucleic acid release solution 15 can be packaged. Based on the pre-packaging of sample processing solution 14 and nucleic acid release solution 15, the sample that needs to be sampled for nucleic acid can be directly added to the sample reaction tube 10 for pre-treatment with sample processing solution 14, and then directly drawn into the rear chamber 6 of the syringe needle 1 to react with nucleic acid release solution 15 to complete the nucleic acid release, making the sample nucleic acid release operation before PCR amplification more convenient and faster. At this time, a ring-shaped retainer 16 that can hold the piston rod 3 in place should also be fitted on the syringe needle 1 (e.g., Figure 2As shown in the figure, an opening slit 17 is also provided on one side of the annular card holder 16 to facilitate removal from the piston push rod 3, thereby effectively preventing the nucleic acid release liquid 15 from being squeezed out of the syringe needle tube 1 due to accidental operation and squeezing of the piston push rod during transfer and transportation.

[0021] In use, simply place the sample to be amplified by PCR (such as swabs, sputum, lavage fluid, etc.) into the sample reaction tube 10, and close the sealing cap 11 to allow it to stand for a period of time to complete the sample pretreatment. Then, pull the piston rod 3 outward to create a negative pressure state in the rear chamber 6 located in front of the rubber piston head. This pressure then draws the first cover plate 8 through the first filter 7, causing the first cover plate 8 to continuously press against and seal the first filter 7. At the same time, the second cover plate 13 will flip clockwise downward to open, thereby drawing the pretreated sample solution from the sample reaction tube 10. After the sample solution passes through the second filter 12 to filter impurities, it enters the rear chamber 6 and reacts with the sample solution in the rear chamber. The nucleic acid release solution 15 in chamber 6 reacts to complete the nucleic acid release; then the piston rod 3 can be pushed inward to squeeze the space of the rear chamber 6 in front of the piston rod 3, so that the second cover plate 13 rotates counterclockwise upward to seal the second filter 12 again. At the same time, the sample solution that has completed the nucleic acid release in the rear chamber 6 will be squeezed through the first filter 7. After the first filter 7 filters out the interfering substances, it pushes the first cover plate 8 to flip up clockwise and open, and then enters the front chamber 5. Finally, it can flow out through the injection port 2 of the syringe needle tube 1, which is connected to the front chamber 5, to realize the operation of adding a small amount of nucleic acid release sample solution to the PCR amplification experimental equipment. The entire device can complete sample pretreatment, nucleic acid release, and filtration in one operation. It is easy to use, low in cost, and highly compatible, and can be applied to the collection and processing of various samples such as swabs, sputum, and lavage fluid. At the same time, it can effectively lyse infectious disease pathogens in the samples to achieve thorough inactivation, reduce inhibitors entering the PCR amplification system, improve the sensitivity and specificity of pathogens in PCR amplification experiments, and enhance the accuracy of pathogen nucleic acid diagnosis. In addition, there is no sample exposure during the entire operation, which greatly improves the biosafety of sample nucleic acid release operations in PCR amplification experiments.

[0022] Example 1:

[0023] This embodiment is a specific example of detecting pulmonary tuberculosis using sputum samples. The sample reaction tube 10 has an outer diameter of 6 cm and an inner diameter of 1 cm at the connection point with the sample inlet 9. The sample reaction tube 10 contains 5 mL of sample processing solution 14, whose main components are 2 mM ethylenediaminetetraacetic acid, 30 mM guanidine isothiocyanate, 200 mM Tris-HCl buffer, and 0.05 mol sodium hydroxide. The rear chamber 6, located in front of the piston rod 3, contains 2 mL of nucleic acid release solution 15. The main components of the discharge solution 15 are 30 mM guanidine isothiocyanate, 200 mM Tris-HCl buffer, 30 mM ammonium sulfate, 2 mM metal ion chelating agent, Triton-100 (v / v) 0.2%, Tween-20 (v / v) 0.2%, ethyl phenyl polyethylene glycol (v / v) 0.15%, sodium dodecyl sulfate (v / v) 0.2%, and sodium lauroyl sarcosinate (v / v) 0.15%. The pore size of the first filter 7 is 0.22 micrometers, and the pore size of the second filter 12 is 0.65 micrometers.

[0024] At this point, add sputum sample to sample reaction tube 10, ensuring the volume ratio of sputum sample to sample processing solution 14 is 1:5. Then, close the sealing cap 11 and let it stand for 10 minutes. After the sample processing solution 14 and sputum sample have fully completed the pretreatment reaction, pull out the piston rod 3 to allow the pretreated sputum sample to be filtered through the second filter 12 and then aspirated into the rear chamber 6 in front of the piston rod 3. Then, gently shake the syringe needle 1 to fully mix the nucleic acid release solution 15 in the rear chamber 6 with the pretreated sputum sample. Let it stand for another 10 minutes to complete the sputum sample reaction. Nucleic acid release; then, keeping the injection port 2 of syringe needle 1 vertically downward, push the piston rod 3 inward, causing the piston rod 3 to move downward and compress the space of the rear chamber 6, thereby squeezing the sputum sample after nucleic acid release through the first filter 7. After the first filter 7 filters out interfering substances, it enters the front chamber 5, and is finally added in a small amount through the injection port 2 to the Mycobacterium tuberculosis complex nucleic acid detection reagent (National Medical Device Registration Certificate 20223401618) for PCR amplification reaction. The nucleic acid amplification equipment used for the PCR amplification reaction is the Applied Biosystems™ 7500 real-time fluorescence quantitative PCR system manufactured by Thermo Fisher Scientific. This system can automatically obtain positive amplification patterns for pulmonary tuberculosis detection (such as... Figure 3 As shown in the figure, curve a is the amplification curve for the detection of Mycobacterium complex nucleic acid, and curve b is the amplification curve for human internal standard. The figure can automatically read the ct value of the amplification result. When the ct value is less than or equal to 40, it indicates that the sputum sample result is determined to be positive for Mycobacterium tuberculosis.

[0025] Example 2:

[0026] This embodiment is a specific example of detecting influenza A using a throat swab sample. The sample reaction tube 10 has an outer diameter of 3 cm and an inner diameter of 1 cm at the connection point with the sample dispensing port 9. The sample reaction tube 10 contains 3 mL of sample processing solution 14, whose main components are 2 mM ethylenediaminetetraacetic acid, 30 mM guanidine isothiocyanate, 200 mM Tris-HCl buffer, and 0.05 mol sodium hydroxide. The rear chamber 6 in front of the piston rod 3 contains 2 mL of nucleic acid release solution 15. The main components of liquid 15 are 20 mM guanidine isothiocyanate, 200 mM Tris-HCl buffer, 30 mM ammonium sulfate, 2 mM metal ion chelating agent, Triton-100 (v / v) 0.15%, Tween-20 (v / v) 0.15%, ethyl phenyl polyethylene glycol (v / v) 0.15%, sodium dodecyl sulfate (v / v) 0.2%, and sodium lauroyl sarcosinate (v / v) 0.15%. The pore size of the first filter 7 is 0.22 micrometers, and the pore size of the second filter 12 is 0.45 micrometers.

[0027] At this point, add the sampled pharyngeal swab sample into the sample reaction tube 10, allowing the pharyngeal swab sample to be immersed in the sample processing solution 14 within the sample reaction tube 10. Then, close the sealing cap 11, manually shake for 1 minute, and let stand for 5 minutes. After the sample processing solution 14 and the pharyngeal swab sample have fully completed the pretreatment reaction, pull out the piston rod 3 to allow the pretreated pharyngeal swab sample solution to be filtered through the second filter 12 and then aspirated into the rear chamber 6 in front of the piston rod 3. Then, gently shake the syringe needle 1 to fully mix the nucleic acid release solution 15 in the rear chamber 6 with the pretreated pharyngeal swab sample solution. Let it stand for another 10 minutes to complete the nucleic acid release of the pharyngeal swab sample solution. After that, keep it in place. Holding the syringe needle 1 with the injection port 2 pointing vertically downwards, the piston rod 3 is pushed inwards, causing the piston rod 3 to move downwards and compress the space of the rear chamber 6. This forces the pharyngeal swab sample solution, after the nucleic acid has been released, through the first filter 7. After the first filter 7 removes interfering substances, the sample enters the front chamber 5 and is finally added in small amounts through the injection port 2 to the influenza A virus nucleic acid detection reagent (National Medical Device Registration Certificate 20173404562) for PCR amplification. The nucleic acid amplification equipment used for the PCR amplification reaction is the SLAN-96P fully automated medical PCR analysis system manufactured by Shanghai Hongshi Medical Technology Co., Ltd. This system can automatically obtain positive amplification patterns for influenza A detection (such as...). Figure 4 As shown in the figure, curve c is the amplification curve for influenza A nucleic acid detection, and curve d is the amplification curve for human internal standard. It can automatically read the ct value of the amplification result. When the ct value is less than or equal to 36, it indicates that the result of the pharyngeal swab sample is positive for influenza A virus.

Claims

1. An integrated sample collection and extraction-free nucleic acid release device, comprising a syringe needle with an injection port at the front end, and a piston rod adapted to be inserted into the syringe needle; characterized in that: The syringe needle tube is divided into a front chamber and a rear chamber by a partition plate. A first filter is provided on the partition plate to connect the front chamber and the rear chamber. A first cover plate for sealing the first filter is hinged to the partition plate on the front chamber side. A sample dispensing port connected to the rear chamber is provided on the syringe needle tube wall. A sample reaction tube extends outward from the sample dispensing port. A sealing cap is fitted to the outer end of the sample reaction tube. A second filter is provided in the sample dispensing port. A second cover plate for sealing the second filter is hinged to the sample dispensing port on the rear chamber side. The hinge point of the second cover plate is located on the side of the sample dispensing port away from the partition plate.

2. The integrated sample collection and extraction-free nucleic acid release device according to claim 1, characterized in that: The sample reaction tube has a flared opening structure.

3. The integrated sample collection and extraction-free nucleic acid release device according to claim 1, characterized in that: The sample reaction tube contains a sample processing solution, and the rear chamber contains a nucleic acid release solution.

4. The integrated sample collection and extraction-free nucleic acid release device according to claim 2, characterized in that: An annular retainer for securing the piston rod is fitted onto the syringe needle, and one side of the annular retainer has an opening for removal from the piston rod.

5. The integrated sample collection and extraction-free nucleic acid release device according to claim 1, characterized in that: Both the first filter and the second filter are disposable needle filters.

6. The integrated sample collection and extraction-free nucleic acid release device according to claim 4, characterized in that: The pore size of the filter membrane of the first filter is 0.45~1 micrometer.

7. The integrated sample collection and extraction-free nucleic acid release device according to claim 4, characterized in that: The filter membrane of the second filter has a pore size of 0.22~0.45 micrometers.

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