An integrated sample detection device and its detection method

Abstract

The present invention provides an integrated sample detection device and a detection method thereof. The device includes a magnetic sleeve and a detection cassette; the detection cassette is provided with at least four side-by-side and non-communicating cavities, and a sealing structure is provided at the top to seal all the cavities. The magnetic sleeve can pierce the sealing structure and can extend into each cavity; among them, the cavities include a lysis cavity for lysing the sample, a magnetic bead cavity pre-loaded with magnetic beads, a washing cavity for washing, and an elution cavity for eluting; a reaction tube is fixed at the bottom of the elution cavity, the top of the reaction tube is sealed, and a detection reagent is pre-loaded in the reaction tube; different reagents for sample extraction are pre-loaded in other cavities except the magnetic bead cavity. The detection cassette of the present invention has a simple structure, low cost, is disposable, has no cross-contamination between samples, and has high safety, providing a broad prospect for the further application of fluorescence quantitative PCR technology in clinical practice.

Description

Technical Field

[0001] The present invention relates to the field of detection technologies, and particularly to an integrated sample detection device and a detection method thereof. Background Art

[0002] The main applications of molecular detection in medical tests include pathogen microorganism infection detection and other aspects. The mainstream technology of molecular detection is fluorescence quantitative PCR technology. Due to the exponential amplification of templates by PCR technology, traditional products mostly use open consumables. For example, in the Chinese patent with publication number CN106442454A and publication date of February 22, 2017: Fluorescence Quantitative Gene Rapid Amplification Detection Device and Amplification Detection Method, the detection kit used in this method is an open consumable. The factors such as aerosol pollution, multiple experimental steps, complex operation, low automation degree, and unstable results easily caused by manual operation brought by open consumables have become important conditions restricting the further application of fluorescence quantitative PCR technology in clinical practice.

[0003] Conventional experimental operations are mostly carried out in an open experimental environment, where samples can be in direct contact with the outside through the air. However, there are often some samples that are highly harmful to operators, easily interfered by the outside or interfere with the outside, such as biological samples containing highly infectious diseases. To reduce biological hazards and at the same time reduce manual operation, there is an urgent need to invent a device for closed sample pretreatment and target substance detection. Summary of the Invention

[0004] The present invention provides an integrated sample detection device and a detection method thereof. The detection device has a simple structure, low cost, is for single use, has no cross - contamination between samples, and has high safety, providing a broad prospect for the further application of fluorescence quantitative PCR technology in clinical practice.

[0005] The technical solution of the present invention is as follows:

[0006] An integrated sample detection device includes a magnetic sleeve and a detection cassette;

[0007] The detection cassette is provided with at least four side - by - side and non - communicating cavities, and a sealing structure is provided at the top to seal all cavities. The magnetic sleeve can pierce the sealing structure and can extend into each cavity;

[0008] Among them, the cavities include a lysis cavity for lysing the sample, a magnetic bead cavity pre - filled with magnetic beads, a washing cavity for washing, and an elution cavity for eluting;

[0009] A reaction tube is fixed at the bottom of the elution cavity, the top of the reaction tube is sealed, and a detection reagent is pre - filled in the reaction tube;

[0010] Except for the magnetic bead cavity, the other cavities are respectively pre - filled with different reagents for sample extraction.

[0011] The detection kit contains multiple cavities, and different cavities can be pre-loaded with different sample extraction reagents; the sealing structure is in close contact with the top of the detection kit to seal the cavities, and the reagents in the cavities are restricted within the cavities; the magnetic sleeve can be inserted into each cavity by an instrument, and the magnetic rod on the instrument can extend into the magnetic sleeve to adsorb the magnetic beads in the reagent. By moving the magnetic beads to different cavities, the material transfer within the cavities can be achieved, and finally the target substance can be transferred to the reaction tube for reaction detection.

[0012] Furthermore, there are two washing cavities, including the first washing cavity and the second washing cavity, to make the sample washing cleaner.

[0013] Furthermore, the magnetic sleeve is a hollow cylindrical tube with a closed conical bottom end and an open top end.

[0014] Furthermore, the reaction tube is a conical sleeve that fits with the bottom end of the magnetic sleeve.

[0015] The magnetic rod can be inserted into the magnetic sleeve through the opening, so that the magnetic sleeve becomes magnetic to adsorb the magnetic beads in the reagent. At the same time, when the conical shape at the bottom end of the magnetic sleeve is inserted into the reaction tube, it can block and seal the reaction tube, further enhancing the airtightness.

[0016] Furthermore, the cross-section of the cavity is one of a triangle, a rectangle, a polygon, an ellipse, and a circle.

[0017] Furthermore, the sealing structure is a sealing film or a sealing cover.

[0018] Furthermore, the sealing film is made of a plastic film or an aluminum film and can be quickly pierced. The sealing film is sealed by heat sealing or ultrasonic welding.

[0019] Furthermore, a tab extends outward from one side of the detection kit, and one end of the sealing film extends to cover the tab. One corner of the sealing film can be torn along the tab to add a detection sample to the lysis cavity, and then the sealing film can be covered again. While facilitating the operation, the sealing performance is enhanced.

[0020] Furthermore, the reaction tube is a PCR tube, and the bottom of the reaction tube is externally connected to a detection instrument.

[0021] The present invention also provides a method for detection using the above-mentioned integrated sample detection device, including the following steps:

[0022] (a) Sample addition: Open the sealing film on the lysis cavity, add the detection sample, then cover the sealing film, and then place the integrated sample detection kit into the detection instrument according to the positioning structure;

[0023] (b) Sample extraction and PCR detection:

[0024] 1) Puncture the sealing film: The detection instrument moves the magnetic sleeve above one of the cavities, and then uses the magnetic sleeve to pierce the sealing film at the corresponding position. Repeat the above steps to pierce the sealing films on all cavities.

[0025] 2) Sample lysis: The detection instrument moves the magnetic sleeve into the lysis cavity, and then moves the magnetic sleeve up and down or rotates it in the cavity to mix and lyse the test sample in the cavity.

[0026] 3) Target capture: The detection instrument moves the magnetic sleeve into the magnetic bead cavity, and moves the magnetic sleeve up and down or rotates it in the cavity to mix the magnetic beads in the cavity; the magnetic rod on the detection instrument is inserted downward into the magnetic sleeve to adsorb the magnetic beads in the cavity and adsorb the magnetic beads onto the magnetic sleeve; the detection instrument moves the magnetic sleeve together with the magnetic rod into the lysis cavity, withdraws the magnetic rod, releases the magnetic beads adsorbed on the magnetic sleeve, and moves the magnetic sleeve up and down in the cavity to mix the reagents and magnetic beads in the cavity, so that the target in the test sample is adsorbed onto the magnetic beads.

[0027] 4) Washing: The magnetic rod of the detection instrument is inserted downward into the magnetic sleeve, and the magnetic sleeve adsorbs the magnetic beads in the lysis cavity; the magnetic sleeve together with the magnetic rod is moved into the washing cavity, the magnetic rod is withdrawn, the magnetic beads adsorbed on the magnetic sleeve are released, and the magnetic sleeve moves up and down in the washing cavity to mix the reagents and magnetic beads in the cavity; then the magnetic beads are transferred to another washing cavity in the same way for secondary cleaning.

[0028] 5) Target elution: The detection instrument moves the magnetic sleeve together with the magnetic rod into the elution cavity, withdraws the magnetic rod, releases the magnetic beads adsorbed on the magnetic sleeve, and moves the magnetic sleeve up and down in the elution cavity to mix the reagents and magnetic beads in the cavity.

[0029] 6) Discard the magnetic beads: The magnetic rod of the detection instrument is inserted downward into the magnetic sleeve to adsorb the magnetic beads in the elution cavity and adsorb the magnetic beads onto the magnetic sleeve; the detection instrument moves the magnetic sleeve together with the magnetic rod into the washing cavity, withdraws the magnetic rod, and discards the magnetic beads adsorbed on the magnetic sleeve in the washing cavity. The target extraction is completed, and the purified target is mixed in the reagent in the elution cavity.

[0030] 7) Target detection: The detection instrument moves the magnetic sleeve into the elution cavity, and then moves the magnetic sleeve downward continuously so that the bottom end of the magnetic sleeve pierces the top of the reaction tube, allowing the reagent containing the target in the elution cavity to flow into the reaction tube below. At this time, the magnetic sleeve blocks and seals the mouth of the reaction tube, and the reagent containing the target is mixed with the detection reagent in the reaction tube. The detection instrument starts the PCR program for detection.

[0031] (c) Device Discarding: After the PCR test is completed, take out the integrated sample test kit from the testing instrument and perform waste biological treatment.

[0032] The beneficial effects of the present invention are as follows:

[0033] The present invention integrates sample pretreatment, separation, extraction, and detection of the target substance in a test kit, enabling the preparation and detection of, for example, nucleic acids or proteins in a closed space without manual sample pretreatment, achieving seamless docking and functional integration throughout the process. The test kit of the present invention greatly simplifies the overall structure, is for single use, has no cross-contamination between samples, reduces the probability of sample contamination, improves the accuracy of sample extraction and detection, can add liquid samples and swab samples, and only requires one-step sample addition operation, which is convenient to use and provides a broad prospect for the further application of fluorescence quantitative PCR technology in clinical practice. Description of the Drawings

[0034] Figure 1 is a schematic structural diagram of the test kit of the present invention;

[0035] In the figure: test kit 1, first cavity 101, second cavity 102, third cavity 103, fourth cavity 104, fifth cavity 105, sixth cavity 106, tab 107, sealing film 2, magnetic beads 3, magnetic sleeve 4, reaction tube 5. Detailed Embodiments

[0036] The drawings are only for illustrative purposes and should not be construed as a limitation of this patent; for better illustration of this embodiment, some components in the drawings will be omitted, enlarged, or reduced, which do not represent the dimensions of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted. The description of the positional relationship in the drawings is only for illustrative purposes and should not be construed as a limitation of this patent.

[0037] Example 1:

[0038] As Figure 1As shown in the figure, an integrated sample detection device includes a magnetic sleeve 4 and a detection box 1. The detection box 1 is made of transparent material. The detection box 1 is provided with six side-by-side and equally high cavities that do not communicate with each other. The cavities are all strip-shaped structures, and a sealing film 2 is provided at the top to seal all the cavities. Among them, the second cavity 102 is a magnetic bead cavity pre-filled with magnetic beads 3. The magnetic sleeve 4 is placed in the third cavity 103. The first cavity 101 is a lysis cavity for lysing the sample. The fourth cavity 104 is the first washing cavity for washing. The fifth cavity 105 is the second washing cavity for washing. The sixth cavity 106 is an elution cavity for elution. Different reagents for sample extraction are pre-filled in the first, fourth, fifth, and sixth cavities respectively. Among them, a reaction tube 5 is fixed at the bottom of the sixth cavity 106. The reaction tube 5 is pre-filled with a detection reagent. The top of the reaction tube 5 is sealed. The reaction tube 5 is a PCR tube, and the bottom of the reaction tube 5 is externally connected to a detection instrument.

[0039] In this embodiment, the magnetic sleeve 4 is a hollow cylindrical tube with a closed bottom end and an open top end. The bottom end of the magnetic sleeve 4 is conical. The reaction tube 5 is a conical sleeve that matches the bottom end of the magnetic sleeve 4. The magnetic bar can be inserted into the magnetic sleeve 4 through the opening, so that the magnetic sleeve 4 has magnetism to adsorb the magnetic beads 3 in the reagent. At the same time, when the conical shape at the bottom end of the magnetic sleeve 4 is inserted into the reaction tube 5, it can block and seal the reaction tube 5, further enhancing the airtightness.

[0040] In this embodiment, the cross-section of the cavity is one of a triangle, a rectangle, a polygon, an ellipse, a circle, or it can also be other irregular shapes, which can be customized according to needs.

[0041] In this embodiment, the sealing film 2 is made of materials such as plastic film or aluminum film and can be quickly pierced. The sealing film 2 is sealed by heat sealing or ultrasonic welding.

[0042] In this embodiment, a tab 107 extends outward from one side of the first cavity 101. One end of the sealing film 2 extends to cover the tab 107. One corner of the sealing film 2 can be torn along the tab 107 to add a detection sample into the first cavity 101, and then the sealing film 2 is covered again, which enhances the sealing performance while facilitating the operation.

[0043] The magnetic sleeve 4 can be moved forward and backward and up and down to the required position through an externally connected detection instrument; after the detection instrument pierces the sealing film 2, the magnetic sleeve 4 is taken out and moved forward and backward to place the magnetic sleeve 4 above the next cavity, and then the magnetic sleeve 4 is moved downward into the cavity. The magnetic sleeve 4 moves up and down or rotates in the cavity to mix the reagents in the cavity. The magnetic rod on the detection instrument is inserted downward into the magnetic sleeve 4 to adsorb the magnetic beads 3 in the cavity; the magnetic sleeve 4 together with the magnetic rod is taken out, moved forward and backward to place the magnetic sleeve 4 above the next cavity, and then the magnetic sleeve 4 together with the magnetic rod is moved downward into the cavity. The magnetic rod is withdrawn to release the magnetic beads 3 adsorbed on the magnetic sleeve 4, and the magnetic sleeve 4 moves up and down in the cavity to mix the reagents in the cavity; repeat the above steps, and transfer the magnetic beads 3 to different cavities to realize the transfer of materials in the cavity, and finally transfer the target to the reaction tube 5 for reaction detection.

[0044] The present invention integrates sample pretreatment and separation, extraction and detection of the target in a detection box, enabling, for example, nucleic acids or proteins to be prepared and detected in a closed space without manual sample pretreatment, realizing seamless docking and functional integration of the whole process. It greatly simplifies the overall structure, is for single use, has no cross-contamination between samples, reduces the probability of sample contamination, and improves the accuracy of sample extraction and detection.

[0045] A method for detection based on the integrated sample detection device described in the above-mentioned Embodiment 1 is provided. The present invention will be further described below by taking nucleic acid extraction and PCR detection as an example. The lysis solution is pre-loaded in the first cavity 101, the washing solution with a lower concentration is pre-loaded in the fourth cavity 104, the washing solution with a higher concentration is pre-loaded in the fifth cavity 105, and the elution solution is pre-loaded in the sixth cavity 106. The specific detection process includes the following steps:

[0046] (a) Sample addition: Open the sealing film 2 on the first cavity 101, add the detection sample, then cover the sealing film 2, and then place the integrated sample detection box into the detection instrument according to the positioning structure;

[0047] (b) Sample extraction and PCR detection:

[0048] 1) Piercing the sealing film 2: The detection instrument pierces the sealing film 2 on the third cavity 103 and extends into the third cavity 103 to take out the magnetic sleeve 4, then moves the magnetic sleeve 4 above another cavity, and then pierces the sealing film 2 at the corresponding position with the magnetic sleeve 4. Repeat the above steps to pierce the sealing film 2 on all cavities;

[0049] 2) Sample lysis: The detection instrument moves the magnetic sleeve 4 into the first cavity 101, and then makes the magnetic sleeve 4 move up and down or rotate in the cavity to mix and lyse the detection sample in the cavity;

[0050] 3) Nucleic acid capture: The detection instrument moves the magnetic sleeve 4 into the second cavity 102, and moves the magnetic sleeve 4 up and down or rotates it in the cavity to mix the magnetic beads 3 in the cavity; the magnetic rod on the detection instrument is inserted downward into the magnetic sleeve 4 to adsorb the magnetic beads 3 in the cavity, and adsorb the magnetic beads 3 onto the magnetic sleeve 4; the detection instrument moves the magnetic sleeve 4 together with the magnetic rod into the first cavity, withdraws the magnetic rod, and releases the magnetic beads 3 adsorbed on the magnetic sleeve 4. The magnetic sleeve 4 moves up and down in the cavity to mix the lysis solution and the magnetic beads 3 in the cavity, so that the target substance in the test sample is adsorbed onto the magnetic beads 3;

[0051] 4) Washing: The magnetic rod of the detection instrument is inserted downward into the magnetic sleeve 4, and the magnetic sleeve 4 adsorbs the magnetic beads 3 in the first cavity 101; the magnetic sleeve 4 together with the magnetic rod is moved into the fourth cavity 104, the magnetic rod is withdrawn, and the magnetic beads 3 adsorbed on the magnetic sleeve 4 are released. The magnetic sleeve 4 moves up and down in the fourth cavity 104 to mix the washing solution and the magnetic beads 3 in the fourth cavity 104; then the magnetic beads 3 are transferred to the fifth cavity 105 in the same way for secondary cleaning;

[0052] 5) Nucleic acid elution: The detection instrument moves the magnetic sleeve 4 together with the magnetic rod into the sixth cavity 106, withdraws the magnetic rod, and releases the magnetic beads 3 adsorbed on the magnetic sleeve 4. The magnetic sleeve 4 moves up and down in the sixth cavity 106 to mix the elution solution and the magnetic beads 3 in the cavity;

[0053] 6) Magnetic bead discard: The magnetic rod of the detection instrument is inserted downward into the magnetic sleeve 4 to adsorb the magnetic beads 3 in the sixth cavity 106, and adsorb the magnetic beads 3 onto the magnetic sleeve 4; the detection instrument moves the magnetic sleeve 4 together with the magnetic rod into the fifth cavity 105, withdraws the magnetic rod, and discards the magnetic beads 3 adsorbed on the magnetic sleeve 4 in the fifth cavity 105. The nucleic acid extraction is completed, and the purified nucleic acid is mixed in the elution solution in the sixth cavity 106;

[0054] 7) Nucleic acid detection: The detection instrument moves the magnetic sleeve 4 into the sixth cavity 106, and then moves the magnetic sleeve 4 downward continuously, so that the bottom end of the magnetic sleeve 4 pierces the top of the reaction tube 5, and the elution solution containing nucleic acid in the sixth cavity 106 flows into the lower reaction tube 5. At this time, the magnetic sleeve 4 blocks and seals the mouth of the reaction tube 5, and the elution solution containing nucleic acid is mixed with the detection reagent in the reaction tube 5. The detection instrument starts the PCR program for nucleic acid detection;

[0055] (c) Device discard: After the PCR detection is completed, take out the integrated sample detection kit from the detection instrument and perform waste biological treatment.

[0056] An integrated sample detection device provided by the present invention reduces the possibility of sample contamination by using disposable consumables, and its device structure is simple, which can reduce the cost of consumables. The present invention can add liquid samples and swab samples, and only requires one-step sample addition operation, which is convenient to use and provides a broad prospect for the further application of the fluorescence quantitative PCR technology in clinical practice.

[0057] Obviously, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. An integrated sample detection device, characterized in that, it includes a magnetic sleeve and a detection box; The detection box is provided with at least four side-by-side and non-communication cavities, and a sealing structure is provided at the top to seal all cavities. The magnetic sleeve can pierce the sealing structure, and the magnetic sleeve can extend into each cavity; Among them, the cavities include a lysis cavity for lysing the sample, a magnetic bead cavity pre-loaded with magnetic beads, a washing cavity for washing, and an elution cavity for eluting; A reaction tube is fixed at the bottom of the elution cavity, and the reaction tube is located inside the elution cavity. The top of the reaction tube is sealed, and the reaction tube is pre-loaded with a detection reagent; Except for the magnetic bead cavity, other cavities are respectively pre-loaded with different reagents for sample extraction; The magnetic sleeve is a hollow cylindrical tube, the bottom end is a closed cone shape, and the top end is open; The reaction tube is a conical sleeve that fits with the bottom end of the magnetic sleeve.

2. The integrated sample detection device according to claim 1, characterized in that, it is provided with two washing cavities, including a first washing cavity and a second washing cavity.

3. The integrated sample detection device according to claim 1, characterized in that, The cross-section of the cavity is one of a triangle, a rectangle, a polygon, an ellipse, and a circle.

4. The integrated sample detection device according to claim 1, characterized in that, The sealing structure is a sealing film or a sealing cover.

5. The integrated sample detection device according to claim 4, characterized in that, The sealing film is made of a plastic film or an aluminum film, and the sealing film is sealed by heat sealing or ultrasonic welding.

6. The integrated sample detection device according to claim 1, characterized in that, A tab extends outward from one side of the detection box, and one end of the sealing structure extends to cover the tab.

7. The integrated sample detection device according to claim 1, characterized in that, The reaction tube is a PCR tube, and the bottom of the reaction tube is externally connected to a detection instrument.

8. A method for detecting using the integrated sample detection device according to any one of claims 1-7 above, characterized in that, it includes the following steps: (a) Sample addition: Open the sealing film on the lysis cavity, add the detection sample, then cover the sealing film, and then place the integrated sample detection box into the detection instrument according to the positioning structure; (b) Sample extraction and PCR detection: 1) Piercing the sealing film: The detection instrument moves the magnetic sleeve above one of the cavities, and then uses the magnetic sleeve to pierce the sealing film at the corresponding position. Repeat the above steps to pierce the sealing films on all cavities; 2) Sample lysis: The detection instrument moves the magnetic sleeve into the lysis cavity, and then moves the magnetic sleeve up and down or rotates in the cavity to mix and lyse the detection sample in the cavity; 3) Target capture: The detection instrument moves the magnetic sleeve into the magnetic bead chamber, moves the magnetic sleeve up and down or rotates it in the chamber to mix the magnetic beads in the chamber; the magnetic force rod on the detection instrument is inserted downward into the magnetic sleeve to adsorb the magnetic beads in the chamber and adsorb the magnetic beads onto the magnetic sleeve; the detection instrument moves the magnetic sleeve together with the magnetic force rod to the lysis chamber, withdraws the magnetic force rod, releases the magnetic beads adsorbed on the magnetic sleeve, and the magnetic sleeve moves up and down in the chamber to mix the reagents and magnetic beads in the chamber, so that the target in the test sample is adsorbed onto the magnetic beads. 4) Washing: The magnetic force rod of the detection instrument is inserted downward into the magnetic sleeve, and the magnetic sleeve adsorbs the magnetic beads in the lysis chamber; the magnetic sleeve together with the magnetic force rod is moved to the washing chamber, the magnetic force rod is withdrawn, the magnetic beads adsorbed on the magnetic sleeve are released, and the magnetic sleeve moves up and down in the washing chamber to mix the reagents and magnetic beads in the chamber; then the magnetic beads are transferred to another washing chamber in the same way for secondary cleaning. 5) Target elution: The detection instrument moves the magnetic sleeve together with the magnetic force rod to the elution chamber, withdraws the magnetic force rod, releases the magnetic beads adsorbed on the magnetic sleeve, and the magnetic sleeve moves up and down in the elution chamber to mix the reagents and magnetic beads in the chamber. 6) Magnetic bead discard: The magnetic force rod of the detection instrument is inserted downward into the magnetic sleeve to adsorb the magnetic beads in the elution chamber and adsorb the magnetic beads onto the magnetic sleeve; the detection instrument moves the magnetic sleeve together with the magnetic force rod to the washing chamber, withdraws the magnetic force rod, and discards the magnetic beads adsorbed on the magnetic sleeve in the washing chamber. The target extraction is completed, and the purified target is mixed in the reagent in the elution chamber. 7) Target detection: The detection instrument moves the magnetic sleeve to the elution chamber, and then moves the magnetic sleeve downward continuously until the bottom end of the magnetic sleeve pierces the top of the reaction tube, so that the reagent containing the target in the elution chamber flows into the lower reaction tube. At this time, the magnetic sleeve blocks and seals the mouth of the reaction tube, and the reagent containing the target is mixed with the detection reagent in the reaction tube. The detection instrument starts the PCR program for detection. (c) Device discard: After the PCR detection is completed, take out the integrated sample detection kit from the detection instrument and perform waste biological treatment.

Images