A novel hybridization capture method and device

Abstract

The present invention provides a novel hybridization capture method and device thereof. A novel hybridization capture device is disclosed, which includes a matching reaction pool assembly, a membrane assembly and an air control assembly. The reaction pool assembly includes a sample loading pool, a plurality of radially arranged reaction pool groups and a waste liquid pool; the membrane assembly is a ductile membrane; the air control assembly includes an inner circle air control channel and an outer circle air control channel. The present invention also discloses a method for hybridization capture using the device. The device of the present invention efficiently and conveniently performs multi-throughput or high-throughput hybridization capture experiments through the clever combination of the upper, middle and lower layers and the orderly arrangement of each reaction pool, maintaining ideal sensitivity, specificity and accuracy. The method of the present invention transfers the hybridization capture technology originally performed on carriers such as ELISA plates and magnetic beads to an integrated device, realizing the miniaturization and automation of the hybridization capture technology, and can simultaneously detect multiple target nucleic acids or multiple samples.

Description

Technical Field

[0001] The present invention belongs to the field of detection devices, and more specifically, relates to a novel hybridization capture method and device thereof. Background Art

[0002] Hybridization capture technology uses antibodies to detect target nucleic acids. The principle is that target DNA hybridizes with an RNA probe (or target RNA hybridizes with a DNA probe) to form a DNA-RNA hybrid. This hybrid acts as an antigen and is recognized and captured by anti-DNA-RNA hybrid antibodies, allowing for detection. Hybridization capture technology does not involve nucleic acid extraction, reverse transcription, or amplification, eliminating the need for expensive PCR amplifiers and a separate nucleic acid amplification laboratory, and therefore requires less operator expertise.

[0003] Typically, hybridization capture technology is implemented on classic solid-phase supports such as ELISA plates and magnetic beads. Hybridization capture technology differs from enzyme-linked immunosorbent assay (ELISA) in that its nucleic acid denaturation and hybridization steps involve temperature fluctuations, and the factors to consider and potential challenges in actual operation are also different.

[0004] The current problems faced by hybridization capture technology are that it is difficult to increase the throughput and the operational convenience needs to be improved. Therefore, such improvements are urgently needed in this field. Summary of the Invention

[0005] The purpose of the present invention is to provide a novel hybridization capture method and device.

[0006] In a first aspect of the present invention, a high-throughput hybridization capture method is provided, comprising:

[0007] (1) A high-throughput hybridization capture device is provided, which includes a matching reaction pool component, a membrane component and an air control component; the reaction pool component includes: a sample addition pool, a plurality of radially arranged reaction pool groups and a waste liquid pool, each column of the reaction pool group includes a reaction pool 1, a reaction pool 2 and a reaction pool 3; the sample addition pool, the reaction pool 1 and the reaction pool 2 are connected in series by a connecting groove; there is a spacing area between the reaction pool 2 and the reaction pool 3, and between the reaction pool 3 and the waste liquid pool; the membrane component is a ductile (elastic) membrane, which is located between the reaction pool component and the air control component; the air control component includes an inner ring air control channel The inner and outer ring air control channels form an air control valve area that cooperates with the inner and outer ring air control channels when the assembly contacts the membrane assembly. In each radially arranged reaction pool group, the interval area between reaction pool 2 and reaction pool 3 corresponds to the air control valve area corresponding to the inner ring air control channel, and the interval area between reaction pool 3 and the waste liquid pool corresponds to the air control valve area corresponding to the outer ring air control channel. The liquid flow in the interval area is controlled by controlling the deformation of the membrane assembly in the air control valve area. When the inner or outer ring air control channels are inflated, the membrane assembly deforms positively (expands), and when the air is evacuated, the membrane assembly deforms negatively (contracts).

[0008] (2) coating the capture antibody against the DNA-RNA hybrid in the reaction pool 3; adding a nucleic acid probe to the reaction pool 2, wherein the nucleic acid probe is an RNA probe when the sample to be tested is DNA, and the nucleic acid probe is a DNA probe when the sample to be tested is RNA;

[0009] (3) adding the sample to be tested to the sample loading pool and introducing it from the sample loading pool to the reaction pool 1; the sample to be tested is a sample that has been subjected to nucleic acid denaturation, or nucleic acid denaturation has been performed in the reaction pool 1;

[0010] (4) Inflate the inner ring air control channel, positively deform the membrane assembly of the air control valve area, and seal the spacer between the reaction pool 2 and the reaction pool 3; introduce the sample to be tested into the reaction pool 2, and hybridize the nucleic acid probe with the corresponding nucleic acid in the sample to form a DNA-RNA hybrid;

[0011] (5) The inner ring air control channel is evacuated, the membrane assembly of the air control valve area is negatively deformed, and the spacer between the reaction pool 2 and the reaction pool 3 is opened; the outer ring air control channel is inflated, the membrane assembly of the air control valve area is positively deformed, and the spacer between the reaction pool 3 and the waste liquid pool is closed; the product after the hybridization reaction is introduced into the reaction pool 3, and the DNA-RNA hybrid is captured with a capture antibody against the DNA-RNA hybrid;

[0012] (6) evacuating the outer ring air control channel, negatively deforming the membrane assembly of the air control valve area, and opening the spacer between the reaction tank 3 and the waste liquid tank; and discharging the waste liquid after the capture reaction into the waste liquid tank;

[0013] (7) Detecting the presence or amount of the captured DNA-RNA hybrid.

[0014] In a preferred embodiment, the number of the plurality of radially arranged reaction cell groups is 2 to 200 groups, such as 5, 10, 12, 15, 20, 30, 50, 80, 100, or 150 groups.

[0015] In another preferred embodiment, in (1), the reaction pool assembly further includes: a positive quality control reaction pool group and a negative quality control reaction pool group independent of the sample addition pool; the positive quality control reaction pool group includes a positive quality control sample addition pool and a positive reaction pool 1 interconnected by a connecting groove, and an independent positive reaction pool 2; there is a spacing area between the positive reaction pool 1 and the positive reaction pool 2, and between the positive reaction pool 2 and the waste liquid pool; the negative quality control reaction pool group includes a negative quality control sample addition pool and a negative reaction pool 1 interconnected by a connecting groove, and an independent negative reaction pool 2; there is a spacing area between the negative reaction pool 1 and the negative reaction pool 2, and between the negative reaction pool 2 and the waste liquid pool; the spacing areas between the positive reaction pool 1 and the positive reaction pool 2, and between the negative reaction pool 1 and the negative reaction pool 2 correspond to the air control valve areas corresponding to the inner circle air control channel, and the spacing areas between the positive reaction pool 2 and the waste liquid pool, and between the negative reaction pool 2 and the waste liquid pool correspond to the air control valve areas corresponding to the outer circle air control channel;

[0016] (2), further comprising: coating a capture antibody against the DNA-RNA hybrid in the positive reaction pool 2 and the negative reaction pool 2, respectively; adding a nucleic acid probe to the positive reaction pool 1 and the negative reaction pool 1, respectively; (3), further comprising: adding a positive quality control and a negative quality control to the positive quality control sample pool and the negative quality control sample pool, respectively; the positive quality control or the negative quality control is a sample subjected to nucleic acid denaturation, or nucleic acid denaturation is performed in the positive quality control sample pool or the negative quality control sample pool;

[0017] (4) After the inner ring air control channel is inflated and the air control valve area membrane assembly is positively deformed, the interval area between the positive reaction pool 1 and the positive reaction pool 2, and between the negative reaction pool 1 and the negative reaction pool 2 is closed; the positive quality control and the negative quality control are introduced into the positive reaction pool 1 and the negative reaction pool 1 respectively to perform hybridization reaction;

[0018] (5) After the inner ring air control channel is evacuated and the air control valve area membrane assembly is negatively deformed, the intervals between the positive reaction pool 1 and the positive reaction pool 2, and between the negative reaction pool 1 and the negative reaction pool 2 are opened (liquid flow can flow); after the outer ring air control channel is inflated and the air control valve area membrane assembly is positively deformed, the intervals between the positive reaction pool 2 and the waste liquid pool, and between the negative reaction pool 2 and the waste liquid pool are closed (liquid flow does not flow); the products after the hybridization reaction are introduced into the positive reaction pool 2 and the negative reaction pool 2, respectively, and the DNA-RNA hybrid is captured with the capture antibody against the DNA-RNA hybrid;

[0019] (6) After the outer ring air control channel is evacuated and the air control valve area membrane assembly is negatively deformed, the interval area between the positive reaction pool 2 and the waste liquid pool, and between the negative reaction pool 2 and the waste liquid pool is opened; the waste liquid after the capture reaction is discharged into the waste liquid pool;

[0020] (7), when detecting the presence or amount of the captured DNA-RNA hybrid, it also includes comparing the detection results with the positive quality control and the negative quality control.

[0021] In another preferred example, the reaction pool assembly further includes: a reagent pool, which is connected to reaction pool 1 of the radially arranged reaction pool group, positive reaction pool 1 of the positive quality control reaction pool group, and negative reaction pool 1 of the negative quality control reaction pool group, for transporting reaction reagents, buffer solutions, or washing reagents; preferably, the reaction reagents include (but are not limited to): detection antibodies against DNA-RNA hybrids; more preferably, they carry detectable markers.

[0022] In another preferred embodiment, in (2), the denaturing reagent is added to the reagent pool and transported to reaction pool 1, positive reaction pool 1 or negative reaction pool 1 to perform nucleic acid denaturation.

[0023] In another preferred embodiment, in (2), the nucleic acid probe is a freeze-dried probe.

[0024] In another preferred embodiment, between (6) and (7), the method further includes the following steps: adding a washing reagent to the reagent pool, and transporting the washing reagent to the reaction pool three, the positive reaction pool two or the negative reaction pool two to remove the uncaptured samples.

[0025] In another preferred embodiment, in (7), when detecting the presence or amount of the captured DNA-RNA hybrid, the detection antibody against the DNA-RNA hybrid is added to the reagent pool and transported to reaction pool three, positive reaction pool two or negative reaction pool two. Preferably, the detection antibody carries a detectable marker; more preferably, it further includes: adding a reagent for detecting the detectable marker (such as a specific substrate of the detectable marker) to the reagent pool and transporting it to reaction pool three, positive reaction pool two or negative reaction pool two, and obtaining the presence or amount of the DNA-RNA hybrid based on the marker.

[0026] In another preferred embodiment, the reaction cell assembly is located at the lower layer of the device, the membrane assembly is located at the middle layer of the device, and the gas control assembly is located at the upper layer of the device.

[0027] In another preferred example, the pool walls of reaction pool 2 and reaction pool 3, the pool walls of reaction pool 3 and waste liquid pool, the pool walls of positive reaction pool 1 and positive reaction pool 2, the pool walls of negative reaction pool 1 and negative reaction pool 2, the pool walls of positive reaction pool 2 and waste liquid pool, and the pool walls of negative reaction pool 2 and waste liquid pool are provided with grooves that are opposite to each other but not connected.

[0028] In another preferred embodiment, in the air control assembly, the inner ring air control channel and the outer ring air control channel are independently connected to the gas supply and extraction device.

[0029] In another preferred embodiment, the reaction cell assembly, membrane assembly and gas control assembly have a central hole at their structural centers; preferably, the central hole matches the centrifugal device (can be sleeved on the centrifugal device).

[0030] In another preferred embodiment, the sample addition pool and / or reagent pool is located in the central area of the device, the multiple rows of reaction pool groups radiate outward from the central area, and the waste liquid pool is located on the periphery of the device.

[0031] In another preferred embodiment, the lower portion of the reaction pool assembly further includes: a temperature control device to adjust the temperature of the reaction or incubation.

[0032] In another preferred embodiment, in the membrane assembly and the gas control assembly, the areas corresponding to the sample addition pool and the reagent pool of the reaction pool assembly are hollow areas.

[0033] In another aspect of the present invention, a device for high-throughput hybridization capture is provided, which includes a matching reaction pool assembly, a membrane assembly and a gas control assembly; the reaction pool assembly includes: a sample addition pool, a plurality of radially arranged reaction pool groups and a waste liquid pool, each column of the reaction pool group includes a reaction pool 1, a reaction pool 2 and a reaction pool 3; the sample addition pool, the reaction pool 1 and the reaction pool 2 are connected in series by a connecting groove; there is a spacer between the reaction pool 2 and the reaction pool 3, and between the reaction pool 3 and the waste liquid pool; the membrane assembly is a ductile (elastic) membrane, which is located between the reaction pool assembly and the gas control assembly; the gas control assembly includes The inner ring air control channel and the outer ring air control channel, after the component contacts with the membrane component, can form an air control valve area that matches the inner ring air control channel and the outer ring air control channel; in each column of radially arranged reaction pool groups, the interval area between reaction pool two and reaction pool three corresponds to the air control valve area corresponding to the inner ring air control channel, and the interval area between reaction pool three and the waste liquid pool corresponds to the air control valve area corresponding to the outer ring air control channel; the liquid flow in the interval area is controlled by controlling the deformation of the membrane component in the air control valve area, and the membrane component is positively deformed (expanded) after the inner ring air control channel or the outer ring air control channel is inflated, and the membrane component is negatively deformed (contracted) after the air is pumped out.

[0034] In a preferred example, the reaction pool assembly also includes: a positive quality control reaction pool group and a negative quality control reaction pool group independent of the sample addition pool; the positive quality control reaction pool group includes a positive quality control sample addition pool and a positive reaction pool one connected to each other by a connecting groove, and an independent positive reaction pool two; there is a spacing area between the positive reaction pool one and the positive reaction pool two, and between the positive reaction pool two and the waste liquid pool; the negative quality control reaction pool group includes a negative quality control sample addition pool and a negative reaction pool one connected to each other by a connecting groove, and an independent negative reaction pool two; there is a spacing area between the negative reaction pool one and the negative reaction pool two, and between the negative reaction pool two and the waste liquid pool; the spacing areas between the positive reaction pool one and the positive reaction pool two, and between the negative reaction pool one and the negative reaction pool two correspond to the air control valve areas corresponding to the inner circle air control channel, and the spacing areas between the positive reaction pool two and the waste liquid pool, and between the negative reaction pool two and the waste liquid pool correspond to the air control valve areas corresponding to the outer circle air control channel.

[0035] In another preferred example, the reaction pool assembly also includes: a reagent pool, which is connected to the reaction pool 1 of the radially arranged reaction pool group, the positive reaction pool 1 of the positive quality control reaction pool group, and the negative reaction pool 1 of the negative quality control reaction pool group, and is used to transport reaction reagents, buffer solutions or washing reagents.

[0036] In another preferred embodiment, the reaction cell assembly is located at the lower layer of the device, the membrane assembly is located at the middle layer of the device, and the gas control assembly is located at the upper layer of the device.

[0037] In another preferred example, the pool walls of reaction pool 2 and reaction pool 3, the pool walls of reaction pool 3 and waste liquid pool, the pool walls of positive reaction pool 1 and positive reaction pool 2, the pool walls of negative reaction pool 1 and negative reaction pool 2, the pool walls of positive reaction pool 2 and waste liquid pool, and the pool walls of negative reaction pool 2 and waste liquid pool are provided with grooves that are opposite to each other but not connected.

[0038] In another preferred embodiment, in the air control assembly, the inner ring air control channel and the outer ring air control channel are independently connected to the gas supply and extraction device.

[0039] In another preferred embodiment, the reaction cell assembly, membrane assembly and gas control assembly have a central hole at their structural centers; preferably, the central hole matches the centrifugal device (can be sleeved on the centrifugal device).

[0040] In another preferred embodiment, the sample addition pool and / or reagent pool is located in the central area of the device, the multiple rows of reaction pool groups radiate outward from the central area, and the waste liquid pool is located on the periphery of the device.

[0041] In another preferred embodiment, the lower portion of the reaction pool assembly further includes: a temperature control device to adjust the temperature of the reaction or incubation.

[0042] In another preferred embodiment, the reaction pool three, the positive reaction pool two or the negative reaction pool two is coated with a capture antibody against DNA-RNA hybrid.

[0043] In another preferred embodiment, the reaction pool 2, the positive reaction pool 1 or the negative reaction pool 1 contains a nucleic acid probe, which is an RNA probe when the sample to be tested is DNA, and is a DNA probe when the sample to be tested is RNA; preferably, the probe is a freeze-dried probe.

[0044] In another aspect of the present invention, there is provided a use of any of the aforementioned devices for performing high-throughput hybridization capture detection.

[0045] In another aspect of the present invention, a detection kit is provided, which includes any of the high-throughput hybridization capture devices described above; preferably, it also includes (but is not limited to): a capture antibody against DNA-RNA hybrids; a nucleic acid probe, which is an RNA probe when the sample to be tested is DNA and a DNA probe when the sample to be tested is RNA; a nucleic acid denaturing reagent (denaturing reagent); a washing reagent; a substrate for recognizing a detectable marker; or a color developer.

[0046] Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 , schematic diagram of the process of conventional hybridization capture technology.

[0048] Figure 2 , a schematic top perspective view of the high-throughput hybridization capture device of the present invention.

[0049] Figure 3 , a schematic diagram of a single set of radially arranged reaction pools and waste liquid pools of the high-throughput hybridization capture device of the present invention.

[0050] Figure 4 , a schematic diagram of the layered structure of the high-throughput hybridization capture device of the present invention, comprising an air control component located at the upper layer, a membrane component located at the middle layer, and a reaction pool component located at the lower layer.

[0051] Figure 5 , schematic diagram of the gas control component of the high-throughput hybridization capture device of the present invention.

[0052] Figure 6 , the membrane component of the high-throughput hybridization capture device of the present invention.

[0053] Figure 7 , the reaction pool assembly of the high-throughput hybridization capture device of the present invention.

[0054] Figure 8 , a schematic diagram of the process of hybridization capture using the high-throughput hybridization capture device.

[0055] The respective reference numerals are as follows:

[0056] 1. Reaction pool assembly (lower layer);

[0057] 11. Sample addition pool;

[0058] 121. Reaction tank 1;

[0059] 122. Reaction tank 2;

[0060] 123. Reaction tank three;

[0061] 13. Waste liquid pool;

[0062] 14. Connecting groove;

[0063] 151. Positive quality control sample pool;

[0064] 152. Positive reaction pool 1;

[0065] 153. Positive reaction pool 2;

[0066] 161. Negative control sample pool;

[0067] 162. Negative reaction pool 1;

[0068] 163. Negative reaction pool 2;

[0069] 17. Reagent pool;

[0070] 2. Membrane assembly (middle layer);

[0071] 3. Air control components;

[0072] 31. Inner ring air control channel;

[0073] 32. Outer ring air control channel;

[0074] 33. Air control valve area;

[0075] 34. Gas supply and extraction device

[0076] 4. Center hole;

[0077] 5. Waste liquid channel. DETAILED DESCRIPTION

[0078] After extensive research, the inventors have developed a novel hybridization capture device and a method for hybridization capture using the device. Through the ingenious combination of upper, middle, and lower layers and the orderly arrangement of reaction cells, the device can efficiently and conveniently perform multi-throughput or high-throughput hybridization capture experiments while maintaining ideal sensitivity, specificity, and accuracy.

[0079] The technical solution of the present invention transfers the hybridization capture technology originally performed on carriers such as ELISA plates and magnetic beads to an integrated device, thereby realizing the miniaturization and automation of the hybridization capture technology and being able to detect multiple target nucleic acids or multiple samples simultaneously.

[0080] the term

[0081] As used herein, the terms "test sample," "test sample," or "test nucleic acid (DNA) sample" are used interchangeably and refer to a nucleic acid sample to be tested, comprising one or more nucleic acids, in which the presence of a target nucleic acid of interest is to be determined. The test sample may be a sample containing double-stranded nucleic acids (before a melting reaction) or a sample containing single-stranded nucleic acids after a melting reaction.

[0082] As used herein, "DNA-RNA hybrid" refers to a nucleic acid comprising two chains, one of which is a DNA chain and the other is an RNA chain, wherein the nucleotide sequences of the DNA chain and the RNA chain are complementary.

[0083] As used herein, a "probe" refers to a single-stranded nucleic acid (preferably RNA in this invention) with a known nucleotide sequence that is substantially complementary to a target nucleic acid and that can form a double strand with the target nucleic acid. The "probe" may or may not carry a fluorescent group. For example, a fluorescent group may be attached to the 5' or 3' end of the probe.

[0084] As used in the present invention, the “radial arrangement” of the reaction cell group means that each reaction cell in a group of reaction cells is arranged along the same axis.

[0085] As used herein, "target nucleic acid (DNA)" refers to a nucleic acid of interest, for example, a nucleic acid associated with HPV virus, or a nucleic acid associated with Candida.

[0086] As used in the present invention, a "capture antibody" refers to an antibody that can be coated on a solid phase carrier and specifically recognizes and binds to the DNA-RNA hybrid, and does not bind to single-stranded nucleic acids (including DNA or RNA, etc.). The "capture antibody" binds to the double-stranded hybrid by recognizing the double helix structure of the double-stranded hybrid, rather than being base sequence specific. Coating antibodies on a solid phase carrier is a technique well known to those skilled in the art. The term "capture antibody" can be used interchangeably with "coating antibody."

[0087] As used herein, a "detection antibody" or "enzyme-conjugated antibody" refers to an antibody that specifically recognizes and binds to the DNA-RNA hybrid and does not bind to single-stranded nucleic acids (including DNA or RNA). The "detection antibody" binds to the double-stranded hybrid by recognizing its double helix structure, rather than being sequence-specific. The "detection antibody" carries a detectable label to report capture of the double-stranded hybrid.

[0088] Hybrid capture device

[0089] The present invention provides a device for high-throughput hybridization capture, which comprises a reaction pool component, a membrane component and a gas control component, which are arranged in the lower, middle and upper layers.

[0090] The reaction pool assembly includes a sample loading pool, multiple radially arranged reaction pool groups, and a waste liquid pool. Each reaction pool group includes reaction pool 1, reaction pool 2, and reaction pool 3. The sample loading pool, reaction pool 1, and reaction pool 2 are connected in series via a connecting groove. A spacer is provided between reaction pools 2 and 3, and between reaction pool 3 and the waste liquid pool. Valves or microstructures with high liquid resistance can be installed at the connections between reaction pools 1, 2, 3, and the waste liquid pool to control the liquid.

[0091] The sample loading reservoir is used to add the sample to be tested. Preferably, the sample loading reservoir is located to one side of the central area of the device. The present invention does not particularly limit the shape of the sample loading reservoir. Taking into account the amount of sample to be tested for hybridization capture, its volume is preferably approximately 0.2-0.4 mL, and its depth is preferably approximately 2-4 mm. However, depending on experimental needs, those skilled in the art may also design the sample loading reservoir to a volume and depth outside of the above ranges, and such designs are also within the scope of the present invention.

[0092] In a preferred embodiment, the device further comprises a reagent pool. Preferably, the sample loading pool is located on the other side of the central area of the device (the opposite side of the sample loading pool). The present invention has no particular restrictions on the shape of the reagent pool. Taking into account the amount of reagent added for hybridization capture, preferably, its volume is about 0.2 to 0.4 mL, and preferably, its depth is about 2 to 4 mm. However, according to experimental needs, those skilled in the art may also design the sample loading pool to a volume and depth outside the range, and such a design is also included in the scope covered by the present invention.

[0093] In the multi-row radially arranged reaction pool group, reaction pool one is connected to the sample addition pool through a connecting groove, so that the sample to be tested can be introduced into reaction pool one, and then introduced into reaction pool two, mixed and reacted with the probe in reaction pool two to obtain a DNA-RNA hybrid.

[0094] In a preferred embodiment, in order to provide a positive quality control product control, the device also includes a positive quality control reaction pool group, including a positive quality control sample pool and a positive reaction pool 1 interconnected by a connecting groove, and an independent positive reaction pool 2; there is a spacer area between the positive reaction pool 1 and the positive reaction pool 2, and between the positive reaction pool 2 and the waste liquid pool.

[0095] In a preferred embodiment, in order to provide a negative quality control product control, the device also includes a negative quality control reaction pool group, including a negative quality control sample pool and a negative reaction pool 1 interconnected by a connecting groove, and an independent negative reaction pool 2; there is a spacer area between the negative reaction pool 1 and the negative reaction pool 2, and between the negative reaction pool 2 and the waste liquid pool.

[0096] The air control component described in the present invention includes an inner ring air control channel and an outer ring air control channel. After the component comes into contact with the membrane component, it can form an air control valve area that cooperates with the inner ring air control channel and the outer ring air control channel; in each column of radially arranged reaction pool groups, the interval area between reaction pool two and reaction pool three corresponds to the air control valve area corresponding to the inner ring air control channel, and the interval area between reaction pool three and the waste liquid pool corresponds to the air control valve area corresponding to the outer ring air control channel; the liquid flow in the interval area is controlled by controlling the deformation of the membrane component in the air control valve area, and the membrane component is positively deformed (expanded) after the inner ring air control channel or the outer ring air control channel is inflated, and the membrane component is negatively deformed (contracted) after the air is pumped out.

[0097] The membrane assembly of the present invention is a ductile (elastic) membrane located between the reaction cell assembly and the gas control assembly. Preferably, the membrane is a thermoplastic elastomer membrane. When air is inflated into the gas control channel above the elastic membrane, the elastic membrane material undergoes positive deformation, closing the gaps between the lower reaction wells and isolating the transfer of liquid between the wells; when air is extracted from the gas control channel above the elastic membrane, the elastic membrane material undergoes negative deformation, increasing the gaps between the lower reaction wells, which is conducive to the transfer of liquid between the wells. When air is not inflated or deflated into the gas control channel above the elastic membrane, the transfer of liquid between the wells can also be achieved under a certain driving force due to the mediation of the membrane.

[0098] Various methods can be used to direct the flow of samples or reagents from the sample reservoir / positive control reservoir / negative control reservoir to the subsequent reaction reservoirs and waste liquid reservoir, including but not limited to pump drive, centrifugal force drive, inflation drive, and suction drive. Preferably, centrifugal force drives the flow of liquid in the device.

[0099] In the present invention, there is no particular restriction on the material for making the reaction cell assembly or the gas control assembly body. Preferably, it is visible and has good mechanical strength. The materials that can be applied include, but are not limited to: acrylic (PMMA, also known as organic glass), polydimethylsiloxane (PDMS) plastics, polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), etc., and can also be glass and metal, etc. In a preferred embodiment of the invention, the assembly is made of PMMA. The transparent property of PMMA can facilitate observation of the reaction state, and the hard property of PMMA can make the assembly not easily deformed, durable and light, and this type of material also has the performance of high temperature resistance (thereby can be used for drying and high temperature sterilization) and moisture resistance (thereby being easy to wash). It should be understood that the present invention is not limited to this type of material, and other materials that can be made into transparent or translucent devices are also available.

[0100] As a preferred embodiment of the present invention, the membrane component uses PDMS (polydimethylsiloxane), which is an elastic material. TPE (thermoplastic elastomer) or similar materials thereof may also be used.

[0101] As a preferred embodiment of the present invention, the reaction cell assembly or the gas control assembly can be obtained by laser, CNC engraving or injection molding; the membrane assembly can be obtained by injection molding or cutting.

[0102] As a preferred embodiment of the present invention, when integrating the three-layer structure, a anti-sticking agent is coated on the structural area corresponding to the gas-controlled valve area in the reaction pool assembly; the upper gas-controlled assembly and the middle membrane assembly are first aligned and hot-pressed to obtain the intermediate layer, and then the intermediate layer and the reaction pool assembly are aligned and hot-pressed to obtain a complete hybridization capture device.

[0103] The device of the present invention controls liquid flow through a clever design. Based on the ductility of the membrane assembly, the device utilizes air inflation (inflation) or air extraction (deflation) of the air-controlled valve area to induce positive or negative deformation in corresponding regions of the membrane assembly, regulating the flow of liquid in the corresponding compartments. This simple and convenient method addresses both the need to seal the reaction vessel during a specific reaction and the need to transfer liquid after a single reaction to the next reaction.

[0104] The present invention also provides a kit containing the hybridization capture device, which includes the high-throughput hybridization capture device described above; preferably, it also includes (but is not limited to): a capture antibody against DNA-RNA hybrids; a nucleic acid probe, which is an RNA probe when the sample to be tested is DNA, and a DNA probe when the sample to be tested is RNA; a nucleic acid denaturing agent (denaturing agent); a washing reagent; a substrate that recognizes the detectable marker; a color developer, etc. Furthermore, the kit may include instructions describing the assembly method of the device of the present invention or specific operating instructions for the hybridization capture method of the present invention.

[0105] Hybrid capture method

[0106] The hybrid capture method of the present invention is based on the basic principle of Figure 1 The process of conventional hybridization capture technology is shown in the figure: when using RNA probes, ① first, double-stranded DNA in the sample is denatured and decomposed into single strands. ② The single-stranded DNA binds to a specific RNA probe to form a DNA-RNA hybrid. ③ The DNA-RNA hybrid binds to a specific anti-DNA-RNA antibody on a solid phase support and is immobilized. ④ It then binds to a labeled specific antibody against the DNA-RNA hybrid. ⑤ The target nucleic acid in the sample is qualitatively detected using the labeled signal. By using different probes, different target nucleic acids can be distinguished.

[0107] When the hybridization capture method is performed using the device, the sample and the denaturing reagent are denatured and melted in reaction pool 1; then they enter reaction pool 2 to mix with the probe reagent therein for hybridization reaction to form a DNA-RNA hybrid; then they enter reaction pool 3, in which anti-DNA-RNA hybrid antibodies are fixed, to capture the hybrid; the remaining liquid enters the waste liquid pool, and then the labeled anti-DNA-RNA hybrid antibodies are injected to form a sandwich complex; preferably, the sample is repeatedly cleaned with a cleaning solution; finally, the labeled signal is qualitatively detected in reaction pool 3.

[0108] By implementing hybridization capture through the device of the present invention, the amount of reagents and samples used can be greatly reduced, thereby lowering reagent costs; the entire process is completed within the same device, which can be automated, avoiding cross-contamination and reducing operations and errors; multiple channels can be integrated on the same device or combined in the form of modules to enable one sample to detect multiple targets, or multiple samples to detect single or multiple targets.

[0109] In the hybridization capture method of the present invention, the denaturation step can be completed within the reaction pool or before adding the sample to the sample pool. That is, in the present invention, the sample to be tested can contain double-stranded nucleic acids or can contain single-stranded nucleic acids after a depolymerization reaction.

[0110] In the hybridization capture method of the present invention, the probe includes an RNA probe and a single-stranded DNA probe. When the sample to be tested is DNA, the nucleic acid probe is an RNA probe; when the sample to be tested is RNA, the nucleic acid probe is a DNA probe.

[0111] As an embodiment of the present invention, the probe may also carry a label, such as a fluorescent group, biotinylation, etc.

[0112] As an embodiment of the present invention, the probe can be freeze-dried and pre-placed in reaction pool 2; or it can be introduced from the sample addition pool after denaturation is completed, at which time reaction pools 1 and 2 can be combined.

[0113] The antibody can be fixed in the reaction pool three by physical adsorption or chemical coupling; it can also be fixed on the surface of magnetic beads, microspheres and other materials first, and then placed in the reaction pool three (pre-placed or injected from the inlet after hybridization).

[0114] The capture antibody and the detection antibody can be prepared using the same or different antibodies. That is, the detection antibody can be the same as or different from the capture antibody, provided it does not carry a detectable label. Unlike proteins, antibodies produced against DNA-RNA hybrids do not require specific sequences or antigenic determinants; they specifically recognize the double-stranded structure of the double-stranded hybrid. Anti-DNA-RNA antibodies (whether monoclonal or polyclonal) can bind to any DNA-RNA double-stranded hybrid. Methods for using specific double-stranded hybrids to prepare antibodies against the double-stranded hybrids are techniques known in the art. For example, polyclonal antibodies can be prepared according to the method of Kitagawa & Stollar (Kitagawa Y, Stollar BD, Mol Immunol 1982, 19: 413-420); or monoclonal antibodies can be prepared according to the method of Fliss et al. (Fliss I, Laurent M, Emond E, et al., Appl Environ Microbiol, 1993, 59 (8): 2698-2705).

[0115] As a preferred embodiment of the present invention, the solution of the detection antibody also contains a certain concentration of NaCl, MgCl2, ZnCl2 and Tris-HCl to ensure that the nonspecific adsorption of the detection antibody is minimized when the detection antibody binds to the DNA-RNA hybrid in a solution containing certain salt ions.

[0116] The detectable label is a reporter molecule attached or conjugated to the detection antibody and used to report the binding status of the detection antibody. Preferably, the detectable label is selected from the group consisting of alkaline phosphatase (AP), horseradish peroxidase (HRP), biotin, avidin, digoxigenin, quantum dots, fluorescent groups (FAM, HEX, CY3, ROX, CY5, etc.), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase, among others. Some detectable labels have specific substrates that, upon contact with the substrate, produce a colorimetric or other detectable or visible reaction, thereby reporting the binding status of the detection antibody. Examples of such substrates include p-nitrophenyl phosphate (p-NPP) and CDP-Star for alkaline phosphatase; o-phenylenediamine (OPD), tetramethylbenzidine (TMB), and ABTS for horseradish peroxidase, among others.

[0117] In an embodiment of the present invention, 14 HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) were detected in a single sample, and the results showed good sensitivity and specificity, with an accuracy of 100%.

[0118] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0119] Materials and methods

[0120] HPV samples: National HPV reference plates, purchased from the China Food and Drug Inspection Institute.

[0121] Preparation of DNA-RNA hybrids (for preparation of anti-DNA-RNA hybrid antibodies): Poly(A) and Poly(dT) were purchased from Sigma (USA). Poly(A) and Poly(dT) were mixed in a solution of 0.15 M NaCl and 0.015 M sodium citrate (pH 7.0) to a concentration of 10 μM. The mixture was incubated at 65°C for 60 minutes to obtain DNA-RNA hybrids.

[0122] Preparation of goat anti-DNA-RNA hybrid antibodies: The DNA-RNA hybrid prepared above was mixed with methylated bovine serum albumin (mBSA) and injected into goats with Complete Freund's Adjuvant. Incomplete Freund's Adjuvant was used for the second and subsequent injections. Goat antibodies were purified using conventional immunopurification methods.

[0123] Mouse anti-DNA-RNA hybrid monoclonal antibody: purchased from Z-BioMed, Inc., USA. The cell line (S9.6) of this antibody was from ATCC, USA (#HB-8730).

[0124] Alkaline phosphatase-labeled anti-DNA-RNA hybrid antibody (AP-coupled antibody): obtained by coupling mouse anti-DNA-RNA hybrid monoclonal antibody with alkaline phosphatase (AP).

[0125] Preparation of probe reagents and quality controls

[0126] The preparation method is as follows:

[0127] (1) First, synthesize the following DNA sequence fragment (SEQ ID NO: 1):

[0128]

[0129] (2) The above synthetic DNA sequence was cloned into the KpnI and XbaI sites of the pUC19 vector to obtain a modified pUC19 vector.

[0130] (3) Genome DNA fragments of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 subtypes were synthesized, and the sequence fragments of each DNA were:

[0131] HPV16: positions 83-1558, 3193-4628, and 5559-7154 in the sequence of GenBank accession number K02718 (three fragments concatenated; the same applies hereinafter);

[0132] HPV18: positions 105–1593, 2376–3867, and 5430–7136 in the sequence of GenBank accession number X05015;

[0133] HPV31: positions 108–1463, 3564–4982, and 5558–7072 in the sequence of GenBank accession number HQ537666;

[0134] HPV33: positions 109-1654, 3789-5087, and 5594-7093 in the sequence of GenBank accession number M12732;

[0135] HPV35: positions 110–1476, 3452–4897, and 5601–7109 in the sequence of GenBank accession number X74477;

[0136] HPV39: positions 107–1608, 2654–4187, and 5643–7160 in the sequence of GenBank accession number M62849;

[0137] HPV45: positions 102–1543, 3421–4872, and 5530–7149 in the sequence of GenBank accession number X74479;

[0138] HPV51: positions 68–1476, 2875–4452, and 5894–7431 in the sequence of GenBank accession number M62877;

[0139] HPV52: positions 89–1565, 2459–4098, and 5565–7154 in the sequence of GenBank accession number X74481;

[0140] HPV56: positions 102–1567, 2432–3896, and 5492–7096 in the sequence of GenBank accession number X74483;

[0141] HPV58: positions 110–1542, 2763–4389, and 5565–7139 in the sequence of GenBank accession number D90400;

[0142] HPV59: positions 55–1398, 3567–4896, and 5606–7132 in the sequence of GenBank accession number X77858;

[0143] HPV66: positions 67–1534, 2679–4231, and 5647–7158 in the sequence of GenBank accession number U31794;

[0144] HPV68: positions 150–1678, 2986–4476, and 5508–7025 in the sequence of GenBank accession number FR751039;

[0145] Furthermore, an NheI site sequence (GCTAGC) was added to the 5' end of the above sequence, and a NotI site sequence (GCGGCCGC) was added to the 3' end.

[0146] (4) The synthesized DNA sequences were cloned into the NheⅠ / NotⅠ sites of the modified pUC19 vector.

[0147] (5) T7 was connected upstream of the above DNA sequence. The constructed vector was used as a template (template DNA), and after digestion with NotⅠ, RNA probes for HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were prepared using T7 RNA polymerase. The reaction conditions for preparing RNA using T7 RNA polymerase were as follows: 10 μl 5× transcription buffer, 10 μl 10 mM 4NTP (ATP, CTP, GTP, UTP) mixture, 1 μg template DNA, 50 U RNase inhibitor, 1.5 μl T7 RNA polymerase (20 U / μl), and DEPC-treated water to a total volume of 50 μl. After the mixture was incubated at 37°C for 100 minutes, 2 μl 0.5 M EDTA was added to terminate the reaction.

[0148] Preparation of quality control products

[0149] Prepare quality control samples containing HPV6 DNA, HPV16 DNA, and HPV58 DNA as follows:

[0150] (1) HPV6 (positions 1-8031 in the sequence of GenBank accession number FR751337), HPV16 (positions 1-7904 in the sequence of GenBank accession number K02718), and HPV58 sequences (positions 1-7824 in the sequence of GenBank accession number D90400) were synthesized by chemical synthesis; KpnⅠ and XbaⅠ sites were added at both ends, respectively.

[0151] (2) The above sequences were cloned into the KpnⅠ and XbaⅠ sites of the pUC18 vector to construct HPV plasmids;

[0152] (3) HPV plasmids were transformed into Escherichia coli DH5α;

[0153] (4) Selecting monoclonal strains on solid agarose plates containing ampicillin resistance;

[0154] (5) The monoclonal strain was expanded in LB medium;

[0155] (6) Extract plasmid DNA and perform identification and quantification. The obtained recombinant plasmid DNA can be used as a quality control product. In the low-risk HPV quality control product, the concentration of HPV6 DNA plasmid is 5pg / ml; in the high-risk HPV high-value quality control product, the concentration of HPV16 DNA plasmid is 5pg / ml, and the concentration of HPV58 DNA plasmid is 5pg / ml; in the high-risk HPV low-value quality control product, the concentration of HPV16 DNA plasmid is 1pg / ml, and the concentration of HPV58 DNA plasmid is 1pg / ml. All are placed independently in each quality control product tube. The quality control product dilution solution also includes: 10mM Tris, 10mM EDTA, pH 7.2±0.2.

[0156] Lyophilization of probes

[0157] Prepare a probe solution containing a freeze-dried protective agent, add a quantitative amount of the solution to the freeze-dried agent, and freeze-dry it using a freeze dryer.

[0158] Example 1: Fabrication of a high-throughput hybridization capture device

[0159] In this embodiment, a device for high-throughput hybridization capture is provided, and its top view is shown as follows: Figure 2 , the schematic diagram of a single group of radially arranged reaction pools and waste liquid pools is as follows Figure 3 , the hierarchical structure diagram is as follows Figure 4 The schematic diagram of its gas control component, membrane component, and reaction pool component is as follows Figures 5-7 .

[0160] according to Figures 2 to 7 The device of the present invention includes a reaction pool component 1, a membrane component 2 and a gas control component 3 that cooperate with each other.

[0161] The reaction pool assembly 1 includes: a sample addition pool 11, multiple rows of radially arranged reaction pool groups and a waste liquid pool 13, each row of reaction pool groups includes reaction pool 1 121, reaction pool 2 122, and reaction pool 3 123; the sample addition pool 11, reaction pool 1 121 and reaction pool 2 122 are connected in series by a connecting groove 14; there are spacing areas (not directly connected) between reaction pool 2 122 and reaction pool 3 123, and between reaction pool 3 123 and waste liquid pool 13.

[0162] The membrane assembly 4 is a membrane with ductility (elasticity) and is located between the reaction cell assembly 1 and the gas control assembly 3 .

[0163] The air control component 3 includes an inner ring air control channel 31 and an outer ring air control channel 32. After the component contacts the membrane component, an air control valve area 33 is formed. The air control valve area 33 expands when gas is introduced through the air control channel and contracts when gas is extracted.

[0164] In each row of radially arranged reaction pool groups, each interval area between reaction pool two 122 and reaction pool three 123 corresponds to the corresponding air-controlled valve area 33 on the inner circle air-controlled channel 31, and the interval area between reaction pool three 123 and the waste liquid pool 13 corresponds to the corresponding air-controlled valve area 33 on the outer circle air-controlled channel 32. The air-controlled valve area 33 changes by controlling the deformation of the membrane assembly 2, thereby controlling the liquid flow in the interval area.

[0165] The reaction pool assembly 1 further includes: a positive quality control reaction pool group and a negative quality control reaction pool group that are independent of the sample addition pool 11 .

[0166] The positive quality control reaction pool group includes a positive quality control sample pool 151 and a positive reaction pool 1 152 interconnected by a connecting groove 14, and an independent positive reaction pool 2 153; there is a spacing area between the positive reaction pool 1 152 and the positive reaction pool 2 153, and between the positive reaction pool 2 153 and the waste liquid pool 13.

[0167] The negative quality control reaction pool group includes a negative quality control sample pool 161 and a negative reaction pool 1 162 interconnected by a connecting groove 14, and an independent negative reaction pool 2 163; there is a spacing area between the negative reaction pool 1 162 and the negative reaction pool 2 163, and between the negative reaction pool 2 163 and the waste liquid pool 13.

[0168] The interval area between the positive reaction pool 1 152 and the positive reaction pool 2 153, and between the negative reaction pool 1 162 and the negative reaction pool 2 163 corresponds to the air control valve area 33 corresponding to the inner circle air control channel 31, and the interval area between the positive reaction pool 2 153 and the waste liquid pool 13, and between the negative reaction pool 2 163 and the waste liquid pool 13 corresponds to the air control valve area 33 corresponding to the outer circle air control channel 32.

[0169] The reaction pool assembly 1 also includes a reagent pool 17, which is connected to the reaction pool 121 of the radially arranged reaction pool group, the positive reaction pool 152 of the positive quality control reaction pool group, and the negative reaction pool 162 of the negative quality control reaction pool group, and is used for transporting reaction reagents, buffer solutions or washing reagents.

[0170] The reaction pool assembly 1 is located at the lower layer of the device, the membrane assembly 2 is located at the middle layer of the device, and the gas control assembly 3 is located at the upper layer of the device.

[0171] The pool wall of reaction pool 2 122 close to reaction pool 3 123, the pool wall of reaction pool 3 123 close to waste liquid pool 13, the pool wall of positive reaction pool 1 152 close to positive reaction pool 2 153, the pool wall of negative reaction pool 1 162 close to negative reaction pool 2 163, the pool wall of positive reaction pool 2 153 close to waste liquid pool 13, and the pool wall of negative reaction pool 2 163 close to waste liquid pool 13 are provided with grooves that are opposite to each other but not connected.

[0172] In the membrane assembly 2 and the gas control assembly 3 , the areas corresponding to the sample addition pool 11 and the reagent pool 17 of the reaction pool assembly are hollow areas.

[0173] In the air control assembly, the inner ring air control channel 31 and the outer ring air control channel 32 are independently connected to the gas supply and extraction device 34.

[0174] The reaction pool assembly 1, membrane assembly 2 and gas control assembly 3 have a central hole 4 at their structural centers. The central hole is a nearly hexagonal hole structure and can be sleeved on a centrifugal device.

[0175] The sample addition pool 11 and the reagent pool 17 are respectively located on both sides of the central hole 4, and multiple rows of reaction pool groups radiate outward from the central area. The waste liquid pool 13 is located on the periphery of the device.

[0176] A waste liquid channel 5 is also provided. After the liquid flowing out of the sample addition pool 11 or the reagent pool 17 is distributed into each reaction pool 121 , the excess liquid directly enters the waste liquid pool 13 through the waste liquid channel 5 .

[0177] To assemble the device, the reaction cell assembly 1, membrane assembly 2, and gas control assembly 3 are first fabricated separately, each matching in shape and size, and then integrated. When integrating the three-layer structure, an anti-sticking agent is applied to the structural area of the reaction cell assembly 1 corresponding to the gas control valve area 33 (to prevent fluid flow from being blocked after bonding). The upper gas control assembly 3 is first aligned and heat-bonded with the middle membrane assembly 2 to form the intermediate layer. The intermediate layer is then aligned and heat-bonded with the reaction cell assembly 1 to complete the hybridization capture device.

[0178] Example 2: Hybridization capture and detection using the device of Example 1

[0179] In this embodiment, the device of Example 1 is used for hybridization capture and detection. The flow chart of hybridization capture using the high-throughput hybridization capture device is as follows: Figure 8 In the figure, (S1) represents liquid inlet sampling (e.g., it can be performed at 12,000 rpm for 12 seconds); (S2) represents hybridization reaction (e.g., it can be performed at 4,000 rpm for 12 seconds); (S3) represents immune reaction (e.g., it can be performed at 5,000 rpm for 30 seconds); and (S4) represents waste liquid discharge (e.g., it can be performed at 4,000 rpm for 10 seconds). The black blocks in the figure indicate the areas where the reaction liquid is located during the corresponding reaction.

[0180] The hybrid capture operation process is as follows:

[0181] (1) Coating the anti-DNA-RNA hybrid antibody in reaction pool three: Coating with goat anti-DNA-RNA hybrid antibody, each well is coated with 100 μl PBS (10 mM phosphate buffer, pH 7.4, 150 mM sodium chloride) containing 0.5 μg / 100 μl goat anti-DNA-RNA hybrid antibody, 4°C, for 16 hours. After coating, wash with PBS three times and then block with PBS containing 3% BSA. 14 kinds of probes are pre-placed in 14 reaction pools two (2.5 ng RNA per well) in a freeze-dried form, and the freeze-dried probe of HPV16 is placed in the reaction pool two of the negative control and positive control channels (there is no reaction pool one in the negative control and positive control channels);

[0182] (2) The denaturation and melting steps were performed outside the device: 200 μL of sample was mixed with 100 μL of 2 M NaOH 5 mM EDTA and incubated at 65°C for 30 min; 20 μL each of negative control (no HPV nucleic acid) and positive control (HPV16 10^5 copies / mL) was mixed with 10 μL of 2 M NaOH 5 mM EDTA and incubated at 65°C for 30 min;

[0183] (3) The sample (melted) and negative and positive quality controls (melted) were added to the sample pool, negative quality control pool, and positive quality control pool, respectively. Centrifuged at 1200 rpm for 12 seconds, and the sample entered the reaction pool 1.

[0184] (4) Inflate the inner air control channel in the upper air control assembly, centrifuge at 4000 rpm for 12 s, and the sample enters the second reaction pool and mixes with the freeze-dried probe inside it;

[0185] (5) The bottom of the second reaction pool is in contact with the heating module, heated to 65°C, and incubated for 1 hour for hybridization;

[0186] (6) After hybridization, the device is separated from the heating module and restored to room temperature. The outer ring air control channel in the upper air control assembly is inflated and the inner ring air control channel is deflated. The device is centrifuged at 5000 rpm for 30 seconds, and the sample enters the reaction pool 3.

[0187] (7) The device was shaken at 1100 rpm for 1 h to complete the capture;

[0188] (8) Deflate the outer ring air control channel, centrifuge at 4000 rpm for 10 s, and drain the waste liquid into the waste liquid pool;

[0189] (9) Add 350 μL of anti-DNA-RNA hybrid antibody labeled with alkaline phosphatase to the reagent pool, inflate the outer ring gas control channel, centrifuge at 5000 rpm for 30 s, and the reagent enters the reaction pool three;

[0190] (10) Standing at room temperature for 45 min to form a sandwich complex;

[0191] (11) The outer ring air control channel is vented, centrifuged at 4000 rpm for 10 s, and the waste liquid is discharged into the waste liquid pool;

[0192] (12) Inject 350 μL of 0.1 M Tris-HCl pH 7.4, 0.6 M NaCl buffer (cleaning solution) into the reagent cell and centrifuge at 4000 rpm for 12 s;

[0193] (13) Repeat step (12) 6 times to wash the unreacted sample and the anti-DNA-RNA hybrid antibody labeled with alkaline phosphatase;

[0194] (14) Add 350 μL of alkaline phosphatase luminescent substrate CDP-Star to the reagent pool, inflate the outer ring gas control channel, centrifuge at 4000 rpm for 12 s, and the substrate reagent enters reaction pool three;

[0195] (15) Incubate at room temperature in the dark for 15 min and read the data using a chemiluminescence analyzer.

[0196] Result determination: If the ratio of the signal value of the positive quality control to the negative quality control is greater than or equal to 2.0, the experimental test is considered valid; if the ratio of the signal value of a certain type of HPV to the negative quality control is greater than or equal to 2.0, the HPV type is considered positive, otherwise it is negative.

[0197] Example 3, sensitivity experiment

[0198] All 14 HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) were mixed at a concentration of 10^5 copies / mL for each type and used as the test sample. Negative quality controls (no HPV nucleic acid) and positive quality controls (10^5 copies / mL) were the same as in Example 2.

[0199] Detection was performed using the hybridization capture and detection method of Example 2. The results are shown in Table 1.

[0200] Table 1

[0201] aisle Number of photons Signal-to-noise ratio Negative quality control 1087 Positive quality control 3369 3.10 16 3424 3.15 18 3295 3.03 31 2930 2.70 33 4011 3.69 35 2855 2.63 39 4218 3.88 45 3030 2.79 51 3680 3.39 52 3763 3.46 56 2897 2.67 58 4689 4.31 59 2980 2.74 66 3498 3.22 68 2907 2.67

[0202] According to the structure in Table 1, each HPV type can obtain a stable positive result at 10^5 copies / mL.

[0203] Example 4, specificity experiment

[0204] Sixteen experiments were conducted using a single HPV type sample at a concentration of 107 copies / mL, including 14 high-risk types and two low-risk types (6 and 11). The results are shown in Tables 2 through 17. Negative controls (no HPV nucleic acid) and positive controls were the same as in Example 2.

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216] According to Tables 2 to 15, each type of HPV virus can show extremely significant positive results (significantly high signal-to-noise ratio) at a sample volume of 10^7 copies / mL; at the same time, no signals were detected in channels of non-corresponding types, and there was no cross-reaction between each type at a sample volume of 10^7 copies / mL.

[0217] According to Tables 16 and 17, when HPV6 (10^7 copies / mL) or HPV11 (10^7 copies / mL) was used as the test sample, negative results were obtained.

[0218] The above results indicate that the accuracy of the test method of the present invention can reach 100%.

[0219] The high-throughput hybridization capture device and detection method of the present invention can detect multiple viruses at one time, meet high-throughput requirements, have stable detection conditions, and save time.

[0220] In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Hangzhou Detong Biotechnology Co., Ltd. <120> A novel hybridization capture method and device <130> 211811 <160> 1 <170> PatentIn version 3.3 <210> 1 <211> 54 <212> DNA <213> Artificial sequence <220> <221> misc_feature <223> polynucleotides <400> 1 gagggtacct aatacgactc actatagggc tagcaaagcg gccgctctag agac 54

Claims

1. A high-throughput hybridization capture method, characterized in that: The method comprises: (1) Providing a high-throughput hybridization capture device, which includes a matching reaction pool component, a membrane component, and a gas control component; The reaction pool assembly includes: a sample addition pool, a plurality of radially arranged reaction pool groups and a waste liquid pool, wherein each row of reaction pool groups includes a reaction pool 1, a reaction pool 2, and a reaction pool 3; the sample addition pool, the reaction pool 1, and the reaction pool 2 are connected in series by a connecting groove; and there is a spacer between the reaction pool 2 and the reaction pool 3, and between the reaction pool 3 and the waste liquid pool; The membrane assembly is a ductile membrane located between the reaction tank assembly and the gas control assembly; The air control component includes an inner ring air control channel and an outer ring air control channel. After the component contacts the membrane component, an air control valve area that matches the inner ring air control channel and the outer ring air control channel can be formed; In each radially arranged reaction pool group, the interval between reaction pool 2 and reaction pool 3 corresponds to the air-controlled valve area corresponding to the inner ring air-controlled channel, and the interval between reaction pool 3 and the waste liquid pool corresponds to the air-controlled valve area corresponding to the outer ring air-controlled channel; the liquid flow in the interval is controlled by controlling the deformation of the membrane assembly in the air-controlled valve area, and the membrane assembly is positively deformed when the inner ring air-controlled channel or the outer ring air-controlled channel is inflated, and negatively deformed when the air is evacuated; (2) coating the capture antibody against the DNA-RNA hybrid in the reaction pool 3; adding a nucleic acid probe to the reaction pool 2, wherein the nucleic acid probe is an RNA probe when the sample to be tested is DNA, and the nucleic acid probe is a DNA probe when the sample to be tested is RNA; (3) adding the sample to be tested to the sample loading pool and introducing it from the sample loading pool to the reaction pool 1; the sample to be tested is a sample that has been subjected to nucleic acid denaturation, or nucleic acid denaturation has been performed in the reaction pool 1; (4) Inflate the inner ring air control channel, positively deform the membrane assembly of the air control valve area, and seal the spacer between the reaction pool 2 and the reaction pool 3; introduce the sample to be tested into the reaction pool 2, and hybridize the nucleic acid probe with the corresponding nucleic acid in the sample to form a DNA-RNA hybrid; (5) The inner ring air control channel is evacuated, the membrane assembly of the air control valve area is negatively deformed, and the spacer between the reaction pool 2 and the reaction pool 3 is opened; the outer ring air control channel is inflated, the membrane assembly of the air control valve area is positively deformed, and the spacer between the reaction pool 3 and the waste liquid pool is closed; the product after the hybridization reaction is introduced into the reaction pool 3, and the DNA-RNA hybrid is captured with a capture antibody against the DNA-RNA hybrid; (6) evacuating the outer ring air control channel, negatively deforming the membrane assembly of the air control valve area, and opening the spacer between the reaction tank 3 and the waste liquid tank; and discharging the waste liquid after the capture reaction into the waste liquid tank; (7) Detecting the presence or amount of the captured DNA-RNA hybrid.

2. The method according to claim 1, wherein (1), the reaction pool assembly further includes: a positive quality control reaction pool group and a negative quality control reaction pool group independent of the sample addition pool; The positive quality control reaction pool group includes a positive quality control sample pool and a positive reaction pool 1, which are interconnected by a connecting groove, and an independent positive reaction pool 2; there are spacers between the positive reaction pool 1 and the positive reaction pool 2, and between the positive reaction pool 2 and the waste liquid pool; The negative quality control reaction pool group includes a negative quality control sample pool and a negative reaction pool 1, which are interconnected by a connecting groove, and an independent negative reaction pool 2; there is a spacer between the negative reaction pool 1 and the negative reaction pool 2, and between the negative reaction pool 2 and the waste liquid pool; The interval area between the positive reaction pool 1 and the positive reaction pool 2, and between the negative reaction pool 1 and the negative reaction pool 2 corresponds to the gas control valve area corresponding to the inner circle gas control channel, and the interval area between the positive reaction pool 2 and the waste liquid pool, and between the negative reaction pool 2 and the waste liquid pool corresponds to the gas control valve area corresponding to the outer circle gas control channel; (2), further comprising: coating a capture antibody against the DNA-RNA hybrid in the positive reaction pool 2 and the negative reaction pool 2, respectively; adding a nucleic acid probe to the positive reaction pool 1 and the negative reaction pool 1, respectively; (3), further comprising: adding a positive quality control and a negative quality control to a positive quality control sample pool and a negative quality control sample pool, respectively; the positive quality control or the negative quality control is a sample subjected to nucleic acid denaturation, or nucleic acid denaturation is performed in the positive quality control sample pool or the negative quality control sample pool; (4) After the inner ring air control channel is inflated and the air control valve area membrane assembly is positively deformed, the interval area between the positive reaction pool 1 and the positive reaction pool 2, and between the negative reaction pool 1 and the negative reaction pool 2 is closed; the positive quality control and the negative quality control are introduced into the positive reaction pool 1 and the negative reaction pool 1 respectively to perform hybridization reaction; (5) After the inner ring air control channel is evacuated and the air control valve area membrane assembly is negatively deformed, the interval between the positive reaction pool 1 and the positive reaction pool 2, and between the negative reaction pool 1 and the negative reaction pool 2 is opened; after the outer ring air control channel is inflated and the air control valve area membrane assembly is positively deformed, the interval between the positive reaction pool 2 and the waste liquid pool, and between the negative reaction pool 2 and the waste liquid pool is closed; the products after the hybridization reaction are introduced into the positive reaction pool 2 and the negative reaction pool 2, respectively, and the DNA-RNA hybrid is captured with the capture antibody against the DNA-RNA hybrid; (6) After the outer ring air control channel is evacuated and the air control valve area membrane assembly is negatively deformed, the interval area between the positive reaction pool 2 and the waste liquid pool, and between the negative reaction pool 2 and the waste liquid pool is opened; the waste liquid after the capture reaction is discharged into the waste liquid pool; (7), when detecting the presence or amount of the captured DNA-RNA hybrid, it also includes comparing the detection results with the positive quality control and the negative quality control.

3. The method according to claim 1 or 2, wherein: The reaction pool assembly also includes: a reagent pool, which is connected to the reaction pool 1 of the radially arranged reaction pool group, the positive reaction pool 1 of the positive quality control reaction pool group and the negative reaction pool 1 of the negative quality control reaction pool group, and is used to transport reaction reagents, buffer solutions or washing reagents.

4. The method according to claim 3, wherein The reaction reagents include: detection antibodies against DNA-RNA hybrids.

5. The method according to claim 4, wherein The detection antibody against the DNA-RNA hybrid carries a detectable label.

6. The method according to claim 3, wherein (2) adding a denaturing reagent to the reagent pool and transporting it to reaction pool 1, positive reaction pool 1 or negative reaction pool 1 to melt the nucleic acid; or (2), wherein the nucleic acid probe is a freeze-dried probe; or Between (6) and (7), the method further includes the steps of: adding a washing reagent to the reagent pool, transporting the washing reagent to the reaction pool 3, the positive reaction pool 2 or the negative reaction pool 2, and removing the uncaptured sample; or (7), when detecting the presence or amount of the captured DNA-RNA hybrid, a detection antibody against the DNA-RNA hybrid is added to the reagent pool and transported to the reaction pool three, the positive reaction pool two or the negative reaction pool two, and the detection antibody carries a detectable marker; a reagent for detecting the detectable marker is added to the reagent pool and transported to the reaction pool three, the positive reaction pool two or the negative reaction pool two, and the presence or amount of the DNA-RNA hybrid is obtained based on the marker.

7. The method according to claim 3, wherein The reaction pool assembly is located at the lower layer of the device, the membrane assembly is located at the middle layer of the device, and the gas control assembly is located at the upper layer of the device; or The pool walls of reaction pool 2 and reaction pool 3, the pool walls of reaction pool 3 and waste liquid pool, the pool walls of positive reaction pool 1 and positive reaction pool 2, the pool walls of negative reaction pool 1 and negative reaction pool 2, the pool walls of positive reaction pool 2 and waste liquid pool, and the pool walls of negative reaction pool 2 and waste liquid pool are provided with grooves that are opposite to each other but not connected; or In the air control assembly, the inner ring air control channel and the outer ring air control channel are independently connected to the gas supply and extraction device; or The reaction pool assembly, membrane assembly and gas control assembly have a central hole in their structural centers; the central hole matches the centrifugal device; or The sample addition pool and / or reagent pool are located in the central area of the device, the multiple rows of radially arranged reaction pool groups radiate outward from the central area, and the waste liquid pool is located on the periphery of the device; or The reaction pool assembly further includes a waste liquid channel, which is connected to the sample pool, the reagent pool, the reaction pool 1, and the waste liquid pool. After the liquid flowing out of the sample pool or the reagent pool is distributed into the reaction pool 1, the excess liquid enters the waste liquid pool through the waste liquid channel. The lower part of the reaction pool assembly also includes: a temperature control device to adjust the temperature of the reaction or incubation.

8. A device for high-throughput hybridization capture, characterized in that: It includes a matching reaction pool component, a membrane component and a gas control component; The reaction pool assembly includes: a sample addition pool, a plurality of radially arranged reaction pool groups and a waste liquid pool, wherein each row of reaction pool groups includes a reaction pool 1, a reaction pool 2, and a reaction pool 3; the sample addition pool, the reaction pool 1, and the reaction pool 2 are connected in series by a connecting groove; and there is a spacer between the reaction pool 2 and the reaction pool 3, and between the reaction pool 3 and the waste liquid pool; The membrane assembly is a ductile membrane located between the reaction tank assembly and the gas control assembly; The air control component includes an inner ring air control channel and an outer ring air control channel. After the component contacts the membrane component, an air control valve area that matches the inner ring air control channel and the outer ring air control channel can be formed; In each radially arranged reaction pool group, the interval between reaction pool 2 and reaction pool 3 corresponds to the air-controlled valve area corresponding to the inner ring air-controlled channel, and the interval between reaction pool 3 and the waste liquid pool corresponds to the air-controlled valve area corresponding to the outer ring air-controlled channel; the liquid flow in the interval is controlled by controlling the deformation of the membrane assembly in the air-controlled valve area, and the membrane assembly is positively deformed when the inner ring air-controlled channel or the outer ring air-controlled channel is inflated, and negatively deformed when the air is evacuated; The reaction pool assembly further includes: a positive quality control reaction pool group and a negative quality control reaction pool group independent of the sample addition pool; The positive quality control reaction pool group includes a positive quality control sample pool and a positive reaction pool 1, which are interconnected by a connecting groove, and an independent positive reaction pool 2; there are spacers between the positive reaction pool 1 and the positive reaction pool 2, and between the positive reaction pool 2 and the waste liquid pool; The negative quality control reaction pool group includes a negative quality control sample pool and a negative reaction pool 1, which are interconnected by a connecting groove, and an independent negative reaction pool 2; there is a spacer between the negative reaction pool 1 and the negative reaction pool 2, and between the negative reaction pool 2 and the waste liquid pool; The interval area between the positive reaction pool 1 and the positive reaction pool 2, and between the negative reaction pool 1 and the negative reaction pool 2 corresponds to the gas control valve area corresponding to the inner circle gas control channel, and the interval area between the positive reaction pool 2 and the waste liquid pool, and between the negative reaction pool 2 and the waste liquid pool corresponds to the gas control valve area corresponding to the outer circle gas control channel; The reaction cell assembly, membrane assembly and gas control assembly have a central hole in their structural center; the central hole matches the centrifugal device; the sample loading pool and / or reagent pool are located in the central area of the device, the multiple rows of radially arranged reaction cell groups radiate outward from the central area, and the waste liquid pool is located on the periphery of the device.

9. The device for high-throughput hybridization capture according to claim 8, wherein: The reaction pool assembly also includes: a reagent pool, which is connected to the reaction pool 1 of the radially arranged reaction pool group, the positive reaction pool 1 of the positive quality control reaction pool group and the negative reaction pool 1 of the negative quality control reaction pool group, and is used to transport reaction reagents, buffer solutions or washing reagents.

10. The device for high-throughput hybridization capture according to claim 8, wherein The reaction pool assembly is located at the lower layer of the device, the membrane assembly is located at the middle layer of the device, and the gas control assembly is located at the upper layer of the device; or The pool walls of reaction pool 2 and reaction pool 3, the pool walls of reaction pool 3 and waste liquid pool, the pool walls of positive reaction pool 1 and positive reaction pool 2, the pool walls of negative reaction pool 1 and negative reaction pool 2, the pool walls of positive reaction pool 2 and waste liquid pool, and the pool walls of negative reaction pool 2 and waste liquid pool are provided with grooves that are opposite to each other but not connected; or In the air control assembly, the inner ring air control channel and the outer ring air control channel are independently connected to the gas supply and extraction device; or The reaction pool assembly further includes a waste liquid channel, which is connected to the sample pool, the reagent pool, the reaction pool 1 and the waste liquid pool. After the liquid flowing out of the sample pool or the reagent pool is distributed into the reaction pool 1, the excess liquid enters the waste liquid pool through the waste liquid channel; or The lower part of the reaction pool assembly further includes: a temperature control device to adjust the reaction or incubation temperature; or The reaction pool 3, the positive reaction pool 2 or the negative reaction pool 2 is coated with a capture antibody against the DNA-RNA hybrid; or The reaction pool 2, the positive reaction pool 1 or the negative reaction pool 1 contains a nucleic acid probe. When the sample to be tested is DNA, the nucleic acid probe is an RNA probe; when the sample to be tested is RNA, the nucleic acid probe is a DNA probe.

11. The device according to claim 10, wherein The probe is a freeze-dried probe.

12. Use of the device according to any one of claims 8 to 11 for high-throughput hybridization capture detection.

13. A detection kit, characterized in that: The device includes the high-throughput hybridization capture device according to any one of claims 8 to 11.

14. The detection kit according to claim 13, wherein It also includes: capture antibodies against DNA-RNA hybrids; A nucleic acid probe, which is an RNA probe when the sample to be tested is DNA, and a DNA probe when the sample to be tested is RNA; Nucleic acid melting reagents; washing reagents; a substrate that recognizes the detectable label; or Color developer.

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