Nucleic acid analysis card box and liquid drop generation system

Abstract

The invention discloses a nucleic acid analysis card box and a liquid drop generation system.The nucleic acid analysis card box comprises a nucleic acid extraction card box, a liquid drop generation vibration needle and an air pump connector, and the liquid drop generation vibration needle and the air pump connector are both communicated with the nucleic acid extraction card box; the nucleic acid extraction card box is used for extracting nucleic acid from a sample; the air pump connector is used for being connected with an air pump to provide pressure for the nucleic acid extraction card box; the liquid drop generating vibration needle generates liquid drops through vibration, swing and precession. The nucleic acid analysis card box comprises the nucleic acid extraction card box for performing nucleic acid extraction on a sample and the liquid drop generation vibration needle for generating liquid drops, so that the structure can give consideration to two structures of nucleic acid extraction and liquid drop generation in the process of quantitatively analyzing DNA or RNA, transfer of nucleic acid extraction is not needed, manual operation steps can be reduced, and the detection efficiency is improved. The nucleic acid pollution risk is reduced, and the turnover efficiency of clinical detection is improved.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic technology, and in particular to a nucleic acid analysis cartridge and a droplet generation system. Background Art

[0002] Digital nucleic acid analysis, such as digital PCR, digital LAMP, digital RPA, etc., is a highly sensitive and accurate molecular biology technology, mainly used for quantitative analysis of DNA or RNA. Compared with traditional nucleic acid amplification analysis methods, digital nucleic acid analysis divides the sample into thousands of tiny reaction units (such as microdroplets or microchambers), so that each reaction unit can be amplified and detected independently, and finally the absolute quantification of the number of nucleic acid targets in the sample to be tested is achieved through Poisson distribution. Existing unit segmentation methods are generally achieved through microdroplets or microchambers, but these methods are heavily dependent on microfluidic chips and lack sample pre-treatment steps.

[0003] Although there are some chip-free micro-reaction unit construction methods (CN104324769B, CN216224451U, CN110066721B), these methods all focus on micro-droplet generation, but ignore the extremely important nucleic acid extraction step. The biological sample to be tested needs to be manually extracted and separated for nucleic acid, and then the purified nucleic acid sample is passed into the droplet generation device to generate a separate reaction unit, which increases the manual operation steps, increases the risk of nucleic acid contamination, and reduces the turnover efficiency of clinical testing.

[0004] Therefore, how to reduce manual operation steps, reduce the risk of nucleic acid contamination, and improve the turnover efficiency of clinical testing during the quantitative analysis of DNA or RNA has become a technical problem that needs to be urgently solved by technical personnel in this field. Summary of the invention

[0005] The present invention proposes a nucleic acid analysis cartridge and a droplet generation system, so as to reduce the manual operation steps, reduce the risk of nucleic acid contamination, and improve the turnover efficiency of clinical testing during the quantitative analysis of DNA or RNA.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present application provides a nucleic acid analysis cartridge, comprising a nucleic acid extraction cartridge, a droplet generation vibration needle, and an air pump connector, wherein the droplet generation vibration needle and the air pump connector are both connected to the nucleic acid extraction cartridge; the nucleic acid extraction cartridge is used to extract nucleic acid from a sample; the air pump connector is used to connect to an air pump to provide pressure for the nucleic acid extraction cartridge; the droplet generation vibration needle generates droplets through vibration, swinging, and precession.

[0008] In some examples, the nucleic acid extraction cartridge includes a cartridge body, a first sealing aluminum foil, a single-sided adhesive tape, a second sealing aluminum foil, and a third sealing aluminum foil, wherein:

[0009] The card box body forms a plurality of chambers connected in sequence to extract nucleic acid from the sample. Each chamber is provided with a reagent filling hole. The chamber located at the upstream end is also provided with a sample addition hole. The chamber located at the downstream end is also provided with a first connection port and a second connection port. The first connection port is used to connect the air pump connector, and the second connection port is used to connect the droplet generation vibration needle. The first sealing aluminum foil is used to seal the sample addition hole; the single-sided tape is used to seal the reagent filling hole; the second sealing aluminum foil is used to seal the first connection port; and the third sealing aluminum foil is used to seal the second connection port.

[0010] In some examples, the sample loading hole is disposed near a left end of the cartridge body, and the first connection port and the second connection port are disposed near a right end of the cartridge body.

[0011] In some examples, the sample loading hole is located on the top surface of the cartridge body.

[0012] In some examples, the first connection port and the second connection port are arranged opposite to each other.

[0013] In some examples, the first connection port is located on the top surface of the card box body, and the second connection port is located on the bottom surface of the card box body.

[0014] In some examples, adjacent chambers are connected via a narrow opening, and a cross-section of the narrow opening gradually decreases from an upstream end to a downstream end.

[0015] In some examples, each chamber is also provided with a vent hole.

[0016] In some examples, the vent hole and the reagent filling hole are both located on the back of the cartridge body, wherein in the height direction, the height of the reagent filling hole is lower than the height of the vent hole, and the single-sided tape seals both the reagent filling hole and the vent hole.

[0017] In some examples, among the plurality of chambers connected in sequence, the reagent in at least one chamber is an organic reagent that is incompatible with the water phase, so as to form an oil-water interface between two adjacent chambers.

[0018] In some examples, seven chambers are arranged in sequence in the cartridge body, which are respectively a lysis chamber, a first oil phase separation chamber, a first cleaning chamber, a second oil phase separation chamber, a second cleaning chamber, a third oil phase separation chamber and an elution chamber from the upstream end to the downstream end;

[0019] There are seven reagent filling holes, namely, a first reagent filling hole, a second reagent filling hole, a third reagent filling hole, a fourth reagent filling hole, a fifth reagent filling hole, a sixth reagent filling hole and a seventh reagent filling hole;

[0020] There are seven ventilation holes, namely, a first ventilation hole, a second ventilation hole, a third ventilation hole, a fourth ventilation hole, a fifth ventilation hole, a sixth ventilation hole and a seventh ventilation hole;

[0021] The first reagent filling hole and the first ventilation hole are connected to the lysis chamber, the second reagent filling hole and the second ventilation hole are connected to the first oil phase separation chamber, the third reagent filling hole and the third ventilation hole are connected to the first cleaning chamber, the fourth reagent filling hole and the fourth ventilation hole are connected to the second oil phase separation chamber, the fifth reagent filling hole and the fifth ventilation hole are connected to the second cleaning chamber, the sixth reagent filling hole and the sixth ventilation hole are connected to the third oil phase separation chamber, and the seventh reagent filling hole and the sixth ventilation hole are connected to the elution chamber.

[0022] In some examples, the card box body further includes an eighth vent hole, which is disposed close to the sample loading hole, and the first sealing aluminum foil seals the sample loading hole and the eighth vent hole simultaneously.

[0023] In some examples, the droplet generation vibrating needle includes an interface adapter, a hose, and a vibrating microneedle, wherein the interface adapter is used to connect to the second connection port, and the hose connects the interface adapter and the vibrating microneedle.

[0024] In some examples, the interface adapter includes a plastic connector and a steel needle, the plastic connector is connected to the second interface, and the steel needle is located inside the plastic connector.

[0025] In the second aspect, the present application also provides another nucleic acid analysis cartridge, comprising a nucleic acid extraction cartridge, a droplet generation vibration needle and an inflation component, wherein the droplet generation vibration needle and the air pump connector are both connected to the nucleic acid extraction cartridge; the nucleic acid extraction cartridge is used to extract nucleic acid from a sample; the inflation component is used to provide pressure to the nucleic acid extraction cartridge; the droplet generation vibration needle generates droplets through vibration, swinging and precession.

[0026] In some examples, the nucleic acid extraction cartridge includes a cartridge body, a first sealing aluminum foil, a single-sided adhesive tape and a third sealing aluminum foil, wherein:

[0027] The card box body forms a plurality of chambers connected in sequence to extract nucleic acid from the sample. Each chamber is provided with a reagent filling hole. The chamber located at the upstream end is also provided with a sample addition hole. The chamber located at the downstream end is also provided with a second connection port, and the second connection port is used to connect the droplet generation vibration needle; the first sealing aluminum foil is used to seal the sample addition hole; the single-sided tape is used to seal the reagent filling hole; and the third sealing aluminum foil is used to seal the second connection port.

[0028] In some examples, the inflation assembly includes a sleeve, a push rod and a buckle, wherein the sleeve is disposed on the card box body and is connected to a cavity located at the downstream end, the push rod is slidably disposed inside the sleeve and applies pressure to the cavity located at the downstream end by moving; the buckle is used to limit the push rod during storage.

[0029] In a third aspect, the present application provides a droplet generation system, comprising a nucleic acid analysis cartridge, a culture dish, and an imaging mechanism, wherein the culture dish contains droplet generation oil, and the nucleic acid analysis cartridge is any of the nucleic acid analysis cartridges described above.

[0030] In a fourth aspect, the present application provides a second droplet generation system, comprising a nucleic acid analysis card box, a strip liquid storage tank and an imaging mechanism, wherein the strip liquid storage tank is a long strip liquid storage tank containing droplet generation oil, and the nucleic acid analysis card box is any of the nucleic acid analysis card boxes as described above.

[0031] In a fourth aspect, the present application provides a droplet generation system, comprising a nucleic acid analysis cartridge, a droplet generation chip and an imaging mechanism, wherein the droplet generation chip is a typical droplet generation chip in the microfluidics field, and the nucleic acid analysis cartridge is any of the nucleic acid analysis cartridges described above.

[0032] It can be seen from the above technical scheme that the nucleic acid analysis cartridge of the present application includes a nucleic acid extraction cartridge for extracting nucleic acid from a sample and a droplet generating vibrating needle for generating droplets. Therefore, this structure can take into account both nucleic acid extraction and droplet generation during the quantitative analysis of DNA or RNA. There is no need to transfer nucleic acid extraction, thereby achieving the purpose of reducing manual operation steps, reducing the risk of nucleic acid contamination, and improving the turnover efficiency of clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some examples or embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work, and the present invention can also be applied to other similar scenarios based on the provided drawings. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

[0034] Figure 1-Figure 3 A schematic diagram of a nucleic acid analysis cartridge provided by an embodiment of the present invention;

[0035] Figure 4a-Figure 5 A schematic diagram of a nucleic acid extraction cartridge provided in an embodiment of the present invention;

[0036] Figure 6-7b A schematic diagram of a droplet generation vibrating needle provided in an embodiment of the present invention;

[0037] Figure 8 A schematic diagram of a nucleic acid extraction cartridge storing reagents provided in an embodiment of the present invention;

[0038] Figure 9a-9gIt is a schematic diagram of the working state of the cartridge in different stages when the nucleic acid analysis cartridge is used for nucleic acid extraction;

[0039] Fig.10 A schematic diagram of a first droplet generation system provided by an embodiment of the present invention;

[0040] Figure 11a-11b A schematic diagram of a second droplet generation system provided by an embodiment of the present invention;

[0041] Fig.12 A schematic diagram of a third droplet generation system provided by an embodiment of the present invention;

[0042] Fig.13 A physical picture of a nucleic acid analysis cartridge provided in an embodiment of the present invention;

[0043] Figure 14-15 A schematic diagram of another nucleic acid analysis cartridge provided in an embodiment of the present invention.

[0044] In the figure, 10-nucleic acid analysis cartridge; 20-culture dish; 30-imaging mechanism; 40-strip liquid storage tank; 50-droplet generation chip;

[0045] 100-nucleic acid extraction cartridge; 200-droplet generation vibration needle; 300-air pump connector; 300'-inflating component;

[0046] 110 - card box body; 120 - first sealing aluminum foil; 130 - single-sided adhesive tape; 140 - second sealing aluminum foil; 150 - third sealing aluminum foil;

[0047] 110a-front side; 110b-back side; 110c-top side; 110d-bottom side; 110e-left side; 110f-right side;

[0048] 1101- lysis chamber; 1102- first oil phase separation chamber; 1103- first cleaning chamber; 1104- second oil phase separation chamber; 1105- second cleaning chamber; 1106- third oil phase separation chamber; 1107- elution chamber; 1108- first narrow opening; 1109- second narrow opening; 1110- third narrow opening; 1111- fourth narrow opening; 1112- fifth narrow opening; 1113- sixth narrow opening; 1114- first connection port; 1115- second connection port;

[0049] 1101a-first reagent filling hole; 1102a-second reagent filling hole; 1103a-third reagent filling hole; 1104a-fourth reagent filling hole; 1105a-fifth reagent filling hole; 1106a-sixth reagent filling hole; 1107a-seventh reagent filling hole;

[0050] 1101b-first vent hole; 1102b-second vent hole; 1103b-third vent hole; 1104b-fourth vent hole; 1105b-fifth vent hole; 1106b-sixth vent hole; 1107b-seventh vent hole;

[0051] 1101c- sample adding hole; 1101d- eighth vent hole;

[0052] 210-interface adapter; 220-hose; 230-vibrating microneedle; 211-plastic connector; 212-steel needle; 2301-plastic interface; 2302-microneedle;

[0053] 310'-sleeve, 320'-push rod; 330'-clip. DETAILED DESCRIPTION

[0054] As described in the background technology, the main purpose of the present invention is to reduce manual operation steps, reduce the risk of nucleic acid contamination, and improve the turnover efficiency of clinical testing during the quantitative analysis of DNA or RNA. The embodiment of the present invention discloses a nucleic acid analysis cartridge, which allows nucleic acid extraction reagents to be stably separated by separation oil through structural design, and the prepared nucleic acid analysis cartridge can be stored or transported at low temperature for a long time.

[0055] See also Figures 1 to 13 The nucleic acid analysis cartridge 10 of the present application may include a nucleic acid extraction cartridge 110, a droplet generation vibration needle 200 and an air pump connector 300, wherein the nucleic acid extraction cartridge is used to extract nucleic acid from a sample; the air pump connector 300 is used to connect to an air pump as a power source for subsequent droplet generation to provide pressure for the nucleic acid extraction cartridge; the droplet generation vibration needle 200 generates droplets by vibration, swinging, precession, and the like.

[0056] The nucleic acid analysis cartridge 10 of the present application includes a nucleic acid extraction cartridge for extracting nucleic acid from a sample and a droplet generation vibrating needle 300 for generating droplets. Therefore, the structure can take into account both nucleic acid extraction and droplet generation during the quantitative analysis of DNA or RNA. There is no need to transfer nucleic acid extraction, thereby achieving the purpose of reducing manual operation steps, reducing the risk of nucleic acid contamination, and improving the turnover efficiency of clinical testing.

[0057] See also Figures 1 to 3 , Figure 1 A three-dimensional view of the nucleic acid extraction cartridge 100, the droplet generation vibration needle 200, and the air pump connector 300 when they are not assembled together is shown; Figure 2 A stereoscopic view of the nucleic acid extraction cartridge 100, the droplet generation vibration needle 200, and the air pump connector 300 when assembled together is shown; Figure 3 An exploded view of the nucleic acid extraction cartridge 100, the droplet generation vibration needle 200, and the air pump connector 300 when not assembled together is shown.

[0058] The nucleic acid extraction cartridge 100 may include a cartridge body 110, a first sealing aluminum foil 120, a single-sided adhesive tape 130, a second sealing aluminum foil 140 and a third sealing aluminum foil 150, wherein:

[0059] The card box body 110 is used to form a plurality of chambers connected in sequence to extract nucleic acid from the sample. Each chamber is provided with a reagent filling hole. The chamber located at the upstream end is also provided with a sample loading hole 1101c. The chamber located at the downstream end is also provided with a first connection port 1114 and a second connection port 1115. The first connection port 1114 is used to connect the air pump connector 300, and the second connection port 1115 is used to connect the droplet generation vibration needle 200; the first sealing aluminum foil 120 is used to seal the sample loading hole 1101c; the single-sided tape 130 is used to seal the reagent filling hole; the second sealing aluminum foil 140 is used to seal the first connection port 1114; and the third sealing aluminum foil 150 is used to seal the second connection port 1115.

[0060] It should be noted that the sample adding hole 1101c is used to add samples, and the reagent filling hole is used to add reagents into the corresponding chamber. According to the sample processing direction, it is divided into an upstream end and a downstream end. For multiple chambers, the chamber used for adding samples is the upstream end, and the downstream end for discharging samples; for each chamber, the end connected to the previous chamber is the upstream end, and the end connected to the next chamber is the downstream end.

[0061] In the above, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0062] Among the multiple chambers connected in sequence, the reagent in at least one chamber is an organic reagent that is incompatible with the water phase, so as to form an oil-water interface between the two adjacent chambers. The chamber filled with the organic reagent becomes an oil phase separation chamber, and the number of the oil phase separation chambers is adjusted according to the operation process of the analysis cartridge.

[0063] When the droplet generation vibration needle 200 and the air pump connector 300 are assembled to the card box body 110, the droplet generation vibration needle 200 pierces the third sealing aluminum foil 150 and communicates with the second connecting port 1115 to realize the air path connection between the droplet generation vibration needle 200 and the nucleic acid extraction card box 100; the air pump connector 300 pierces the second sealing aluminum foil 140 and communicates with the first connecting port 1114 to realize the air path connection between the air pump connector 300 and the nucleic acid extraction card box 100.

[0064] The shape of the card box body 110 in this application is not specifically limited. In order to facilitate sealing, the shape of the card box body 110 in this application is a rectangular parallelepiped structure. For ease of understanding, the side facing the reader is the front side 110a, and the side opposite to the front side 110a is the back side 110b. In the height direction, it has a relative top surface 110c and a bottom surface 110d, and in the length direction, it has a relative left side 110e and a right side 110f. Of course, the above definition of the surface is only defined by the orientation represented by the view. Here, the height direction, the length direction and the width direction are perpendicular to each other, and the height direction corresponds to the height of the card box body 110, the length direction corresponds to the length of the card box body 110, and the width direction corresponds to the width of the card box body 110.

[0065] In order to optimize the operation, the sample adding end and the droplet generating end are separated by a preset distance, wherein the sample adding end is used for adding samples, and the droplet generating end is used for generating droplets. In some examples of the present application, the sample adding hole 1101c is arranged near one end of the left side 110e, and the first connecting port 1114 and the second connecting port 1115 are arranged near one end of the right side 110f.

[0066] In order to optimize the above operation, the sample adding hole 1101c is located on the top surface 110c and close to one end of the left surface 110e. When adding samples, they are added from top to bottom, which complies with the principle of gravity and is easy to operate.

[0067] The first connection port 1114 and the second connection port 1115 are arranged relative to each other, which can reduce the gas resistance from the first connection port 1114 to the second connection port 1115. Furthermore, in order to ensure the stability of droplet formation, the first connection port 1114 is located on the top surface 110c of the card box body 110, and the second connection port 1115 is located on the bottom surface 110d of the card box body 110, so that the gas enters from the top of the card box body 110 and is formed from the bottom.

[0068] In order to further optimize the above solution, the reagent filling hole is arranged away from the sample adding hole 1101c. The reagent filling hole is located on the front surface 110a, the back surface 110b, and the bottom surface 110d. The front surface 110a and the back surface 110b are preferably selected.

[0069] See also Figure 4a , Figure 4b , Figure 4c and Figure 5 , Figure 4a A perspective view of the cartridge body 110 is shown; Figure 4b Shown Figure 4a Sectional view of section AA; Figure 4c Shown Figure 4a Sectional view of the middle BB section; Figure 5 A cross-sectional view of the cartridge body 110 is shown.

[0070] In the figure, the cartridge body 110 is provided with seven chambers connected in sequence, from the upstream end to the downstream end (from left to right), respectively, a lysis chamber 1101, a first oil phase separation chamber 1102, a first cleaning chamber 1103, a second oil phase separation chamber 1104, a second cleaning chamber 1105, a third oil phase separation chamber 1106 and an elution chamber 1107, wherein:

[0071] The lysis chamber 1101 is used to add lysis solution, magnetic beads and other reagents.

[0072] The first oil phase separation chamber 1102 , the second oil phase separation chamber 1104 , and the third oil phase separation chamber 1106 are used to add organic reagents incompatible with the water phase, such as mineral oil, paraffin oil, and low melting point paraffin.

[0073] The first cleaning chamber 1103 and the second cleaning chamber 1105 are used to add cleaning solutions required for nucleic acid extraction.

[0074] The elution chamber 1107 is used to add the elution solution required for nucleic acid extraction.

[0075] Each chamber is correspondingly provided with a reagent filling hole. Specifically, there are seven reagent filling holes, namely the first reagent filling hole 1101a, the second reagent filling hole 1102a, the third reagent filling hole 1103a, the fourth reagent filling hole 1104a, the fifth reagent filling hole 1105a, the sixth reagent filling hole 1106a, and the seventh reagent filling hole 1107a; the first reagent filling hole is connected to the lysis chamber 1101, the second reagent filling hole 1102a is connected to the first oil phase separation chamber 1102, the third reagent filling hole 1103a is connected to the first cleaning chamber 1103, the fourth reagent filling hole 1104a is connected to the second oil phase separation chamber 1104, the fifth reagent filling hole 1105a is connected to the second cleaning chamber 1105, the sixth reagent filling hole 1106a is connected to the third oil phase separation chamber 1106, and the seventh reagent filling hole 1107a is connected to the elution chamber 1107.

[0076] The first oil phase separation chamber 1102, the second oil phase separation chamber 1104, and the third oil phase separation chamber 1106 can form an oil-water interface between the first oil phase separation chamber 1102 and the cracking chamber 1101, and between the first oil phase separation chamber 1102 and the first cleaning chamber 1103; the second oil phase separation chamber 1104 can form an oil-water interface between the second oil phase separation chamber 1104 and the first cleaning chamber 1103, and between the second oil phase separation chamber 1104 and the second cleaning chamber 1105; the third oil phase separation chamber 1106 can form an oil-water interface between the third oil phase separation chamber 1106 and the second cleaning chamber 1105, and between the third oil phase separation chamber 1106 and the elution chamber 1107. This allows the reagents in the corresponding chambers to be stably stored.

[0077] In order to improve the smoothness of filling the reagent, each chamber is also provided with a vent. Under the premise that the cartridge body 110 includes seven chambers, there are seven vents, namely, the first vent 1101b, the second vent 1102b, the third vent 1103b, the fourth vent 1104b, the fifth vent 1105b, the sixth vent 1106b, and the seventh vent 1107b. The first reagent filling hole and the first air vent 1101b are in communication with the lysis chamber 1101, the second reagent filling hole 1102a and the second air vent 1102b are in communication with the first oil phase separation chamber 1102, the third reagent filling hole 1103a and the third air vent 1103b are in communication with the first cleaning chamber 1103, the fourth reagent filling hole 1104a and the fourth air vent 1104b are in communication with the second oil phase separation chamber 1104, the fifth reagent filling hole 1105a and the fifth air vent 1105b are in communication with the second cleaning chamber 1105, the sixth reagent filling hole 1106a and the sixth air vent 1106b are in communication with the third oil phase separation chamber 1106, and the seventh reagent filling hole 1107a and the sixth air vent 1106b are in communication with the elution chamber 1107.

[0078] It should be noted that in order to reduce the number of sealing steps, the reagent filling holes and the vent holes are both located on the back side 110b, wherein the height of the reagent filling holes is lower than the height of the vent holes in the height direction. The single-sided adhesive 130 can seal the reagent filling holes 1101a-1107a and the vent holes 1101b-1107b at the same time.

[0079] Similarly, in order to improve the smoothness of sample addition, the cartridge body 110 may further include an eighth vent hole 1101d, which is connected to the outside during the sample addition process. In order to reduce the number of sealing steps, the eighth vent hole 1101d is arranged close to the sample addition hole 1101c, and the first sealing aluminum foil 120 may be used to simultaneously seal the sample addition hole 1101c and the eighth vent hole 1101d.

[0080] Adjacent chambers are connected by narrow openings, and the cross section of the narrow openings gradually decreases from the upstream end to the downstream end, that is, it is in a contracted shape. The various liquids in the cartridge form an oil-water interface at the narrow openings of adjacent chambers, and remain stable under the surface tension of the oil-water two phases, and can withstand a certain degree of disturbance. On the premise that the card box body 110 includes seven chambers, there are six narrow openings, which are, from left to right, the first narrow opening 1108, the second narrow opening 1109, the third narrow opening 1110, the fourth narrow opening 1111, the fifth narrow opening 1112 and the sixth narrow opening 1113. The first narrow opening 1108 connects the lysis chamber 1101 with the first oil phase separation chamber 1102, the second narrow opening 1109 connects the first oil phase separation chamber 1102 with the first cleaning chamber 1103, the third narrow opening 1110 connects the first cleaning chamber 1103 with the second oil phase separation chamber 1104, the fourth narrow opening 1111 connects the second oil phase separation chamber 1104 with the second cleaning chamber 1105, the fifth narrow opening 1112 connects the second cleaning chamber 1105 with the third oil phase separation chamber 1106, and the sixth narrow opening 1113 connects the third oil phase separation chamber 1106 with the elution chamber 1107.

[0081] When filling the reagents, firstly, the sample loading hole 1101c, the vent hole 1101d, the first connection port 1114, and the second connection port 1115 of the cartridge body 110 are sealed with the first sealing aluminum foil 120, the second sealing aluminum foil 140, and the third sealing aluminum foil 150 respectively; the lysis solution, magnetic beads and other reagents required for nucleic acid extraction are added to the lysis chamber 1101 through the reagent filling port 1101a; mineral oil, paraffin oil, low melting point paraffin and other organic reagents incompatible with the water phase are added to the first oil phase separation chamber 1102 through the reagent filling port 1102a; the cleaning solution required for nucleic acid extraction is added to the first cleaning chamber 1103 through the reagent filling port 1103a; mineral oil, paraffin oil, low melting point paraffin and other organic reagents incompatible with the water phase are added to the first oil phase separation chamber 1102 through the reagent filling port 1103a. Incompatible organic reagents are added to the second oil phase separation chamber 1104 through the reagent filling port 1104a; the cleaning solution required for nucleic acid extraction is added to the second cleaning chamber 1105 through the reagent filling port 1105a; mineral oil, paraffin oil, low melting point paraffin and other organic reagents incompatible with the aqueous phase are added to the third oil phase separation chamber 1106 through the reagent filling port 1106a; the eluent required for nucleic acid extraction is added to the elution chamber 1107 through the reagent filling port 1107a, where the eluent is a nucleic acid amplification reagent that can perform an amplification reaction, such as a LAMP reaction system, a PCR reaction system, etc.; finally, the back side 110b of the card box body 110 is sealed with a single-sided tape 130 to complete the pre-filling of the reagents. At this point, the nucleic acid extraction card box 100 containing the nucleic acid extraction reagent can be stored or transported for a long time at low temperature, and the state of each liquid in the card box body 110 is as follows Figure 8 As shown, the black color is the oil phase organic reagent and the gray color is the water phase reagent.

[0082] See also Figure 6 , Figure 7a and Figure 7b , Figure 6 An exploded view of the droplet generation vibrating needle 200 is shown, Figure 7a A front view of a droplet generation vibrating needle 200 is shown; Figure 7b Shown Figure 7a Cross-sectional view of section CC.

[0083] In the figure, the droplet generation vibrating needle 200 may include an interface adapter 210 , a hose 220 and a vibrating microneedle 230 . The interface adapter 210 is used to connect to the second connection port 1115 , and the hose 220 connects the interface adapter 210 and the vibrating microneedle 230 .

[0084] The interface adapter 210 may include a plastic connector 2101 and a steel needle 2102, wherein the plastic connector 2101 is used to connect to the second connection port 1115, and the steel needle 2102 is arranged at one end of the plastic connector 2101 close to the second connection port 1115, and is used to puncture the third sealing aluminum foil 150. The hose 220 has good flexibility and can be bent; the vibrating microneedle 230 may include a plastic interface 2301 and a microneedle 2302, wherein the outer diameter of the microneedle is about 20 microns and the inner diameter is about 10 microns. When in use, the steel needle 2102 punctures the third sealing aluminum foil 150, and realizes the liquid path connection between the droplet generation vibration needle 200 and the nucleic acid extraction cartridge 100.

[0085] See also Figure 8 , Figure 8 The state of the nucleic acid extraction cartridge 100 when storing various reagents is shown. The black color is the oil phase organic reagent separating the various water phases, and the gray color is the water phase nucleic acid extraction reagent. The oil and water phases exist stably due to the design of the narrow openings 1108-1113.

[0086] See also Figure 9a-9g , Figure 9a-9g The process of nucleic acid extraction using the magnetic bead method using the nucleic acid extraction cartridge 100 is demonstrated.

[0087] First, use the tip of a pipette to pierce the first sealed aluminum foil 120, add the sample into the lysis chamber 1101 through the sample loading hole 1101c, and discharge the excess air through the vent 1101d. Then, use a single-sided adhesive to seal the sample loading hole 1101c and the vent 1101d. Then, connect the nucleic acid extraction cartridge body 110 to the air pump connector 300 and the droplet generation vibration needle 200 respectively. The air pump connector 300 should be an adapter component of the air pump, which is connected to the air pump. At this time, the air pump does not provide pressure, and this is a dead end. Therefore, the liquid in the elution chamber 1107 can exist stably and will not flow out from the droplet generation vibration needle 200. Figure 9a shown.

[0088] Then, the magnet is placed against the front side 110a of the cartridge body 110, which faces the lysis chamber 1101. The magnetic beads enriched with nucleic acid will gather on the inner wall of the lysis chamber 1101. Figure 9b shown.

[0089] Then, under the action of the magnet, it slowly passes through the first narrow opening 1108 and enters the first oil phase separation chamber 1102. Fig.9c shown.

[0090] Then, the magnet is continuously moved to allow the magnetic beads enriched with nucleic acid to enter the first cleaning chamber 1103 through the second narrow opening 1109 for magnetic bead cleaning. Figure 9d shown.

[0091] Then, the magnet is continuously moved to allow the magnetic beads enriched with nucleic acid to pass through the third narrow opening 1110, the second oil phase separation chamber 1104, and the fourth narrow opening 1111 into the second cleaning chamber 1105 for a second cleaning of the magnetic beads. Fig.9e shown.

[0092] Then, the magnet is continuously moved to allow the magnetic beads enriched with nucleic acid to enter the elution chamber 1107 through the fifth narrow opening 1112, the third oil phase separation chamber 1106, and the sixth narrow opening 1113 for nucleic acid elution. The eluted nucleic acid is fully mixed with the amplification system. Figure 9f shown.

[0093] Finally, the magnetic beads are enriched by a magnet and pass through the sixth narrow opening 1113, the third oil phase separation chamber 1106, and the fifth narrow opening 1112 into the second cleaning chamber 1105 at one time. Figure 9g shown.

[0094] See also Fig.10 , Fig.10 A schematic diagram of the first droplet generation system is shown.

[0095] The droplet generation system includes a nucleic acid analysis cartridge 10, a culture dish and an imaging mechanism, wherein the culture dish 20 contains droplet generation oil, and the vibrating microneedle 230 penetrates 0-1mm into the oil phase liquid surface. At this time, the external air pump provides pressure, and the amplification system in the elution chamber 1107 flows out from the vibrating microneedle 230 through the droplet generation vibrating needle 200 under the action of air pressure. The droplet generation vibrating needle 200 swings left and right, vibrates left and right, or precesses around the rotation center under the action of an external driving force. At this time, the amplification system flowing out of the vibrating microneedle 230 is subjected to the shear force generated by the droplet generation oil that is stationary in the culture dish 20, and is discrete into droplets and settles at the bottom of the culture dish 20 and spreads flat. Here, the droplet generation oil can be a mineral oil containing a surfactant, isopropyl palmitate, or a solidifiable oil phase that is heated under a Pt catalyst and subjected to a silylation reaction. After the droplet generation is completed, the culture dish 20 containing the droplets is heated on a heating device to perform a nucleic acid amplification reaction, and the nucleic acid amplification reaction can be RPA, LAMP or PCR, etc. Finally, the culture dish 20 after the reaction is subjected to fluorescence imaging and moved to the imaging mechanism 30 for detection, and the original target content is calculated by Poisson distribution.

[0096] See also Fig.11a and Fig.11b , Fig.11a and Fig.11b A schematic diagram of the second droplet generation system is shown.

[0097] The droplet generation system includes a nucleic acid analysis cartridge 10, a strip liquid reservoir 40 and an imaging mechanism, wherein the strip liquid reservoir 40 is a long strip liquid reservoir containing droplet generation oil, the vibrating microneedle 230 penetrates 0-1 mm into the oil phase liquid surface, and the strip liquid reservoir 40 is placed on the surface of a temperature-controllable heating device, which has two temperature zones, a low temperature zone (below room temperature) and a high temperature zone (39°C or 65°C). At this time, the external air pump provides pressure, and the amplification system in the elution chamber 1107 flows out from the vibrating microneedle 230 through the droplet-generating vibrating needle 200 under the action of air pressure. The droplet-generating vibrating needle 200 swings left and right, vibrates left and right, or precesses around the center of rotation under the action of an external driving force. The amplification system flowing out of the vibrating microneedle 230 is subjected to the shear force generated by the stationary droplet-generating oil in the strip liquid reservoir 40, and is dispersed into droplets and settled flat on the bottom of the strip liquid reservoir 40. At the same time, the strip liquid reservoir 40 is slowly pushed from left to right, and the strip liquid reservoir 40 gradually enters the high-temperature area from the low-temperature area. When the droplets are heated in the high-temperature area to reach the reaction temperature, the internal amplification system begins to perform a nucleic acid amplification reaction. As the strip liquid reservoir 40 gradually moves, it enters the detection area of ​​the imaging mechanism 40, such as Fig.11bAs shown. By setting appropriate movement time and droplet generation speed, a pipeline working mode of simultaneous generation and detection of micro-droplet nucleic acid detection can be realized. The detection process is similar to the detection of punched paper tape. The droplets are regarded as through holes in the punched paper tape, and the negative and positive droplets in the strip storage tank 40 are continuously counted. It is suitable for simultaneous generation, amplification and detection of a large number of droplets. The droplet generation oil here can be a mineral oil containing a surfactant, isopropyl palmitate, or a solidifiable oil phase that is heated under a Pt catalyst and undergoes a hydrosilylation reaction.

[0098] See also Fig.12 , Fig.12 A schematic diagram of the third droplet generation system is shown.

[0099] The droplet generation system includes a nucleic acid analysis cartridge 10, a droplet generation chip 50 and an imaging mechanism, wherein the droplet generation chip 50 is a typical droplet generation chip in the microfluidics field, and micro droplets are generated inside the droplet generation chip by means of T-tubes, flow focusing, coaxial flow, etc. At this time, the 230 microneedles of the nucleic acid analysis cartridge 10 are inserted into the droplet generation chip as an aqueous phase input, the external air pump of the nucleic acid analysis cartridge 10 is used as a driving source, and another inlet of the droplet generation chip is used as an oil phase input, with additional droplet generation oil and a corresponding driving source. The generated droplets can be amplified and detected in situ in the chip, or transferred to an EP tube for amplification and detection after being collected. The droplet generation oil here can be a mineral oil containing a surfactant, isopropyl palmitate, or a solidifiable oil phase heated under a Pt catalyst through a hydrosilylation reaction.

[0100] See also Fig.13 , Fig.13 This is a physical picture of the nucleic acid extraction cartridge 100 of the present invention. The dark part is dyed nucleic acid extraction related aqueous phase reagent, and the transparent liquid between the dark parts is mineral oil used to separate the aqueous phase.

[0101] See also Fig.14 and Fig.15 , Fig.14 A three-dimensional view of another nucleic acid analysis cartridge 10 is shown; Fig.14 An exploded view of another nucleic acid analysis cartridge 10 is shown.

[0102] The nucleic acid analysis cartridge 10 shown in the figure is Figures 1 to 3The difference of the nucleic acid analysis cartridge 10 shown is that the structure for providing pressure has changed. In this example, the structure for providing pressure is that the inflation component 300' may include a sleeve 310', a push rod 320' and a buckle 330'. The sleeve 310' is arranged on the cartridge body 110 and communicates with the cavity at the downstream end. The push rod 320' is slidably arranged inside the sleeve 310' and applies pressure to the elution cavity 1107 by moving; the buckle 330' is used to limit the push rod 320' during storage to prevent the push rod 320' from changing its position due to bumps and shaking, etc., which affects the internal pressure balance.

[0103] By slowly pushing the push rod 320', pressure can be applied to the elution chamber 1107, so that the liquid in the elution chamber is slowly discharged, achieving the same effect as an external air pump. The buckle 330' is used to limit the push rod 320' during storage to prevent the push rod 320' from changing its position due to bumps and shaking, which affects the internal pressure balance. The above design makes the nucleic acid analysis cartridge 10 more integrated, reduces gas line connections, reduces the risk of nucleic acid contamination, and reduces the requirements for supporting instruments. Its specific implementation method is not essentially different from the aforementioned cartridge.

[0104] The above description is only a preferred embodiment of the present invention and an explanation of the technical principles used, and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. The scope of the invention involved in the present invention is not limited to the technical solution formed by a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with the technical features with similar functions disclosed in the present invention (but not limited to) to form a technical solution.

Claims

1. A nucleic acid analysis cartridge, characterized in that: It includes a nucleic acid extraction card box, a droplet generation vibration needle and an air pump connector, wherein the droplet generation vibration needle and the air pump connector are both connected to the nucleic acid extraction card box; the nucleic acid extraction card box is used to extract nucleic acid from a sample; the air pump connector is used to connect to an air pump to provide pressure for the nucleic acid extraction card box; the droplet generation vibration needle generates droplets through vibration, swinging and precession.

2. The nucleic acid analysis cartridge according to claim 1, wherein: The nucleic acid extraction card box comprises a card box body, a first sealing aluminum foil, a single-sided adhesive tape, a second sealing aluminum foil and a third sealing aluminum foil, wherein: The card box body forms a plurality of chambers connected in sequence to extract nucleic acid from the sample, each of the chambers is provided with a reagent filling hole, the chamber located at the upstream end is also provided with a sample addition hole, and the chamber located at the downstream end is also provided with a first connection port and a second connection port, the first connection port is used to connect the air pump connector, and the second connection port is used to connect the droplet generation vibration needle; the first sealing aluminum foil is used to seal the sample addition hole; the single-sided tape is used to seal the reagent filling hole; the second sealing aluminum foil is used to seal the first connection port; and the third sealing aluminum foil is used to seal the second connection port.

3. The nucleic acid analysis cartridge according to claim 2, wherein: The sample addition hole is arranged near one end of the left side of the cartridge body, and the first connection port and the second connection port are arranged near one end of the right side of the cartridge body.

4. The nucleic acid analysis cartridge according to claim 3, wherein: The sample addition hole is located on the top surface of the cartridge body.

5. The nucleic acid analysis cartridge according to claim 3, wherein: The first connection port and the second connection port are arranged opposite to each other.

6. The nucleic acid analysis cartridge according to claim 5, characterized in that: The first connection port is located on the top surface of the card box body, and the second connection port is located on the bottom surface of the card box body.

7. The nucleic acid analysis cartridge according to claim 5, wherein: Adjacent chambers are connected via narrow openings, and the cross-section of the narrow openings gradually decreases from the upstream end to the downstream end.

8. The nucleic acid analysis cartridge according to claim 5, wherein: Each of the chambers is also provided with a vent hole.

9. The nucleic acid analysis cartridge according to claim 5, wherein: The vent hole and the reagent filling hole are both located on the back side of the cartridge body, wherein in the height direction, the height of the reagent filling hole is lower than the height of the vent hole, and the single-sided adhesive seals the reagent filling hole and the vent hole simultaneously.

10. The nucleic acid analysis cartridge according to claim 2, wherein: Among the plurality of chambers connected in sequence, the reagent in at least one chamber is an organic reagent that is incompatible with the water phase, so as to form an oil-water interface between two adjacent chambers.

11. The nucleic acid analysis cartridge according to claim 10, wherein: The cartridge body is provided with seven chambers connected in sequence, which are respectively a lysis chamber, a first oil phase separation chamber, a first cleaning chamber, a second oil phase separation chamber, a second cleaning chamber, a third oil phase separation chamber and an elution chamber from the upstream end to the downstream end; There are seven reagent filling holes, namely, a first reagent filling hole, a second reagent filling hole, a third reagent filling hole, a fourth reagent filling hole, a fifth reagent filling hole, a sixth reagent filling hole and a seventh reagent filling hole; There are seven ventilation holes, namely, a first ventilation hole, a second ventilation hole, a third ventilation hole, a fourth ventilation hole, a fifth ventilation hole, a sixth ventilation hole and a seventh ventilation hole; The first reagent filling hole and the first ventilation hole are in communication with the lysis chamber, the second reagent filling hole and the second ventilation hole are in communication with the first oil phase separation chamber, the third reagent filling hole and the third ventilation hole are in communication with the first cleaning chamber, the fourth reagent filling hole and the fourth ventilation hole are in communication with the second oil phase separation chamber, the fifth reagent filling hole and the fifth ventilation hole are in communication with the second cleaning chamber, the sixth reagent filling hole and the sixth ventilation hole are in communication with the third oil phase separation chamber, and the seventh reagent filling hole and the sixth ventilation hole are in communication with the elution chamber.

12. The nucleic acid analysis cartridge according to claim 11, wherein: The card box body also includes an eighth vent hole, which is arranged close to the sample addition hole, and the first sealing aluminum foil seals the sample addition hole and the eighth vent hole at the same time.

13. The nucleic acid analysis cartridge according to claim 2, wherein: The droplet generation vibrating needle comprises an interface adapter, a hose and a vibrating microneedle, wherein the interface adapter is used to be connected to the second connection port, and the hose connects the interface adapter and the vibrating microneedle.

14. The nucleic acid analysis cartridge according to claim 13, wherein: The interface adapter includes a plastic connector and a steel needle. The plastic connector is connected to the second interface, and the steel needle is located inside the plastic connector.

15. A nucleic acid analysis cartridge, characterized in that: It includes a nucleic acid extraction card box, a droplet generation vibration needle and an inflation component, wherein the droplet generation vibration needle and the air pump connector are both connected to the nucleic acid extraction card box; the nucleic acid extraction card box is used to extract nucleic acid from a sample; the inflation component is used to provide pressure to the nucleic acid extraction card box; the droplet generation vibration needle generates droplets through vibration, swinging and precession.

16. The nucleic acid analysis cartridge according to claim 15, wherein: The nucleic acid extraction card box comprises a card box body, a first sealing aluminum foil, a single-sided adhesive and a third sealing aluminum foil, wherein: The card box body forms a plurality of chambers connected in sequence to extract nucleic acid from the sample, each of the chambers is provided with a reagent filling hole, the chamber located at the upstream end is also provided with a sample addition hole, and the chamber located at the downstream end is also provided with a second connection port, and the second connection port is used to connect the droplet generation vibration needle; the first sealing aluminum foil is used to seal the sample addition hole; the single-sided tape is used to seal the reagent filling hole; and the third sealing aluminum foil is used to seal the second connection port.

17. The nucleic acid analysis cartridge according to claim 16, wherein: The inflation assembly includes a sleeve, a push rod and a buckle, wherein the sleeve is arranged on the card box body and is connected to the cavity located at the downstream end, the push rod is slidably arranged inside the sleeve, and applies pressure to the cavity located at the downstream end by moving; the buckle is used to limit the push rod during storage.

18. The nucleic acid analysis cartridge according to claim 16, wherein: Among the plurality of chambers connected in sequence, the reagent in at least one chamber is an organic reagent that is incompatible with the water phase, so as to form an oil-water interface between two adjacent chambers.

19. A droplet generation system, characterized in that: It comprises a nucleic acid analysis cartridge, a culture dish and an imaging mechanism, wherein the culture dish contains droplet generation oil, and the nucleic acid analysis cartridge is the nucleic acid analysis cartridge as claimed in any one of claims 1 to 18.

20. A droplet generation system, characterized in that: It comprises a nucleic acid analysis card box, a strip liquid storage tank and an imaging mechanism, wherein the strip liquid storage tank is a long strip liquid storage tank containing droplet generation oil, and the nucleic acid analysis card box is the nucleic acid analysis card box as described in any one of claims 1 to 18.

21. A droplet generation system, characterized in that: It comprises a nucleic acid analysis cartridge, a droplet generation chip and an imaging mechanism, wherein the droplet generation chip is a typical droplet generation chip in the microfluidics field, and the nucleic acid analysis cartridge is the nucleic acid analysis cartridge as claimed in any one of claims 1 to 18.

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