CN116925903A

CN116925903A - Nucleic acid detection system

Info

Publication number: CN116925903A
Application number: CN202211107997.1A
Authority: CN
Inventors: 金霞; 刘杰; 胡彬; 肖杰; 黄富强
Original assignee: Hangzhou Xunling Biotechnology Co ltd
Current assignee: Hangzhou Xunling Biotechnology Co ltd
Priority date: 2022-01-11
Filing date: 2022-09-09
Publication date: 2023-10-24
Also published as: CN220685130U; CN117143715A; CN220413390U

Abstract

The application provides a nucleic acid detection system, which integrates the functions of sample adding, nucleic acid purification, isothermal amplification and immunochromatography for reading detection results on a device, and the PEG and other reagents in a nucleic acid amplification reagent, such as a recombinase reagent and the like, are separated and then are dried respectively, are mixed when reacted, and improve the formulas of lysate and eluent, so that the influence on the nucleic acid amplification reaction can be prevented, the detection sensitivity can be improved, the operation process is simplified, and the omission and the false detection are prevented; the prepared nucleic acid detection device is simple and small, low in cost and simple and convenient to operate, and can finish nucleic acid detection through a twice rotation mode; the method is lower in cost when used for detecting nucleic acid, simpler and more convenient to operate, high in detection sensitivity, short in required time and applicable to detecting nucleic acid of various samples such as human, animals and the like.

Description

Nucleic acid detection system

The application claims priority of China prior application, application number 2022100300570, application day 2022, 1 month 11; all of which are included as part of the present application.

Technical Field

The application belongs to the technical field of biological detection, and relates to a nucleic acid detection system.

Background

With the development of science and technology, detection technologies and equipment in the fields of pathogens, environmental microorganisms, biochemical indexes, tumor, organ markers and the like are greatly developed. Such as PCR instruments and isothermal amplification instruments applying the nucleic acid amplification principle, the traditional smear or microscopic examination method is gradually replaced in the pathogen and environmental microorganism detection fields; the automatic chemiluminescence platform provides a sensitive, rapid and high-flux solution for detection projects such as biochemical indexes, tumor markers and the like.

The expansion of detection requirements and the enrichment of detection scenes make POCT (Point-of-care testing) type detection one of the main directions of the development of the detection field in recent years. POCT (point of care testing) refers to a testing form for instantly analyzing at a sampling site and rapidly obtaining a test result. For example, POCT nucleic acid detector integrates all the functions of nucleic acid extraction, amplification, fluorescence detection and the like into a miniaturized instrument, and can perform rapid and accurate nucleic acid detection under non-laboratory conditions.

Compared with the traditional large-scale equipment or standard laboratory, the POCT detection equipment provides a field detection means for vast basic units, remote areas or other scenes with insufficient medical conditions. Meanwhile, the method provides a convenient and fast scheme for detection items which are sensitive to detection time. Many POCT products such as small blood glucose meters, urine glucose test strip devices, etc. have begun to walk into home settings where the detection is accomplished by the user in a self-test format.

In many cases, it is desirable to be able to perform the detection by means of self-test; under the conditions of some field environments or lack of professionals and equipment, people are more urgent to need a detection device which is low in cost, quick, simple, accurate in result and capable of being used without professional training. The device should have a wide range of scene adaptations and safety in use.

However, the current small-sized detection device still has a plurality of problems: 1. the current miniaturized detection instrument still has the problem of high cost for common people; 2. because nucleic acid detection often needs to be carried out for nucleic acid amplification or collection and analysis of fluorescent signals, relatively complex equipment and professional skills are required, and at present, the equipment for domestic self-detection cannot realize the nucleic acid detection yet; 3. although the existing test paper self-checking products meet certain requirements, the problems of inaccurate sample adding, difficulty in quantitative and multiple detection and the like still exist; 4. the detection sensitivity of the current self-checking device is low; 5. the current self-checking device needs longer time for detection; 6. the existing home detection device lacks flexibility and has the problems of repeated development and resource waste.

CN113512490a provides a self-driven microfluidic detection device, and sample loading, amplification and detection are realized through a microfluidic diversion component, but the microfluidic diversion process is easily affected by ambient temperature and humidity, and detection errors are easily generated. WO2022/043697A1 provides a device for analysing biological samples, but it requires selective opening of different areas by means of control means such as tie rods, pistons, etc. to effect transfer of the sample from one area to another, with cumbersome operation, error-prone and complex structure.

In addition, the existing nucleic acid detection devices directly adopt the existing detection reagents such as lysate, nucleic acid amplification reaction reagent, eluent and the like, but do not carry out related research on whether the reagents have influence on each other, so that the problems of low detection sensitivity, missed detection, false detection and the like are caused.

Therefore, the market needs a disposable detection device which has low cost, simple structure, rapidness, convenience, high detection sensitivity, short detection time, safety and reliability, can be used without professional training and is matched with the disposable detection device, so that the detection sensitivity is improved, and the problems of missing detection, false detection and the like are prevented.

Disclosure of Invention

In order to make up the defects of the prior art, the invention provides a nucleic acid detection system, which comprises a nucleic acid detection device and a reagent, wherein proper lysate and eluent are screened to prevent the nucleic acid amplification reaction from being influenced, PEG and enzyme in the nucleic acid amplification reagent are separated and then are dried respectively, and are mixed when reacted, so that the sensitivity of nucleic acid detection is obviously improved, and omission and false detection are prevented.

The nucleic acid detection system provided by the invention comprises a nucleic acid detection device and a matched nucleic acid detection reagent, wherein the functions of sample adding, nucleic acid purification, isothermal amplification and immunochromatography for reading detection results can be all integrated on one device, a lysate is added into a sample, the lysed sample is added through a sample adding port, nucleic acid in the sample is adsorbed through a nucleic acid adsorption film, and redundant sample enters a filter paper storage tank; then washing the nucleic acid in the nucleic acid adsorption film to a reaction cavity through eluent to perform isothermal amplification reaction, wherein a fixed and dried nucleic acid amplification reagent is stored in the reaction cavity in advance; and transferring the amplified product to a test strip for detection. The nucleic acid detection device prepared by the invention is simple and small, has lower cost, simpler and more convenient operation, high detection sensitivity and short time when being used for detecting nucleic acid, and can be used for detecting nucleic acid of various samples such as human, animals and the like.

In one aspect, the invention provides a nucleic acid detection system comprising a nucleic acid detection device and reagents including a lysate, an eluate, and a nucleic acid amplification reaction reagent; the nucleic acid amplification reaction reagent is a reagent after drying and immobilization and comprises a first immobilization reagent and a second immobilization reagent, wherein the first immobilization reagent contains enzyme, and the second immobilization reagent contains PEG.

The nucleic acid amplification reaction reagent at least comprises recombinase, primer probe combination, DNTP, ATP, dithiothreitol, PEG and Mg ²⁺ Etc., wherein PEG is used to provide a reaction environment, mg ²⁺ Is an initiator for isothermal amplification reaction.

In order to make the detection process more convenient and flexible, the invention places and fixes the dry nucleic acid amplification reaction reagent in the nucleic acid detection device in advance, thereby avoiding the need of adding a plurality of reagents from the sample adding port for many times in the detection process and preventing the reagent from being adsorbed by the nucleic acid adsorption film of the sample adding port to influence the isothermal amplification reaction and leading to detection errors.

Because the nucleic acid amplification reaction reagent contains complex components, it is necessary to consider whether the immobilized reagent can be stored in the reaction chamber for a long period of time after the extraction, immobilization and drying treatment, and the nucleic acid amplification reaction will not be affected after the reconstitution. In order to achieve good amplification effect, PEG is added into the nucleic acid amplification reaction reagent, the molecular weight of the PEG is preferably 20000-40000, and a great amount of researches prove that the PEG and other reagents in the nucleic acid amplification reaction reagent, such as recombinase and the like, need to be dried and fixed separately, otherwise, the isothermal amplification reaction of nucleic acid can be seriously influenced, so that the detection sensitivity is influenced, and the reason is probably that the PEG can shrink and wrap the enzyme in the drying process, and the enzyme activity is difficult to recover after later re-dissolution, so that the isothermal amplification reaction of nucleic acid cannot be normally carried out.

Further, the first immobilized reagent also contains a primer probe, a single-chain binding protein, a protein cofactor and DNTP.

In some embodiments, the second immobilizing reagent may further comprise magnesium acetate.

The primer probe may be co-dried with the enzyme and solidified, but not dried with the PEG, probably because PEG also affects the concentration of the primer probe.

Further, the nucleic acid detection device comprises a sample processing cavity, a sample reaction cavity and a detection cavity which are sequentially arranged from top to bottom; the processing sample cavity is used for adsorbing nucleic acid in a sample; the sample reaction cavity is used for completing nucleic acid amplification reaction; the detection chamber is used for detecting nucleic acid in the amplified product.

Further, the sample reaction cavity comprises a reaction cavity for performing a nucleic acid amplification reaction; the nucleic acid amplification reaction reagent is placed in the reaction chamber of the sample reaction chamber in advance.

Further, the nucleic acid amplification reaction reagent is an immobilization reaction membrane or a dry reagent.

The immobilized reaction membrane adopts membranes made of silica gel, glass microfiber filter paper filter membranes and the like, the membrane thickness is about 1mm, the shape is round or any other shape, preferably a round membrane with the diameter of 6mm, the nucleic acid amplification reaction reagent can be fixed on the membrane in a drying, freeze-drying and other modes, and the temperature cannot exceed 50 ℃ during drying.

Further, when the nucleic acid amplification reaction reagent is an immobilized reaction membrane, the immobilized reaction membrane comprises a first reaction membrane and a second reaction membrane, wherein the first reaction membrane contains a first immobilized reagent, and the second reaction membrane contains a second immobilized reagent; when the nucleic acid amplification reaction reagent is a dry reagent, the dry reagent includes a first dry reagent containing a first immobilization reagent and a second dry reagent containing a second immobilization reagent; when the nucleic acid amplification reaction reagent is a dry reagent, a parting bead is required to be arranged at the bottom of the reaction cavity, and the first dry reagent and the second dry reagent are separated by the parting bead.

In some embodiments, the dried reagent may be prepared by oven drying, freeze drying, at a temperature not exceeding 50 ℃.

In some modes, the parting bead at the bottom of the reaction cavity can be in the form of a parting sheet like the middle part of a double-taste hot pot, so long as the effect of separating two reagents can be achieved, the two reagents can be respectively added into two sides of the parting bead in the reaction cavity, and then freeze-drying or drying is carried out, so that the nucleic acid detection device with the preselected nucleic acid amplification reaction reagent can be prepared.

In some modes, the height of the parting bead at the bottom of the reaction cavity cannot be too high, generally is 2mm, only two reagents can be separated in advance, and when isothermal amplification is carried out after re-dissolution, the two reagents still need to be mixed together for reaction, so that the situation that the parting bead is too high to cause mixing is prevented.

Further, the lysate includes Tris (hydroxymethyl) aminomethane (Tris), ethylenediamine tetraacetic acid, guanidine isothiocyanate, and triton 100.

After the nucleic acid sample cracked by the lysate is adsorbed by the nucleic acid adsorption film, although the redundant sample enters the filter paper storage tank, the lysate still remains on the nucleic acid adsorption film, and then the nucleic acid sample is washed into the reaction cavity together by the eluent to perform the nucleic acid isothermal amplification reaction. Therefore, the components in the lysate cannot influence the isothermal amplification reaction of the nucleic acid, and cannot influence the detection process of the nucleic acid, otherwise, the detection sensitivity of the nucleic acid is influenced, and the detection is missed or misprimed.

The lysate provided by the invention does not have adverse effect on nucleic acid amplification reaction, so that the target nucleic acid can be eluted by using the eluent and enter isothermal amplification reaction without adding a cleaning agent for flushing after the nucleic acid in the sample is adsorbed by the nucleic acid adsorption film, and the lysate is particularly suitable for the nucleic acid detection device provided by the invention.

Further, the processing sample cavity comprises a nucleic acid adsorption membrane, wherein the nucleic acid adsorption membrane is a silica GF/C membrane or a silica gel membrane.

Further, the eluent includes magnesium acetate, ethylenediamine tetraacetic acid and Tris (hydroxymethyl) aminomethane (Tris).

After the eluent is added from the sample adding port, the nucleic acid adsorbed by the nucleic acid adsorption film can be eluted into the reaction cavity for isothermal amplification reaction.

Mg ²⁺ The initiator for the isothermal amplification reaction may be added to the eluent for eluting the nucleic acids, and added together with the elution of the nucleic acids from the nucleic acid adsorption membraneIn the reaction cavity, the nucleic acid elution effect is not affected, and the isothermal nucleic acid amplification reaction can be smoothly started.

The eluent provided by the invention can be used for efficiently eluting nucleic acid into the reaction cavity, starting the isothermal amplification reaction of the nucleic acid and effectively improving the detection sensitivity of the nucleic acid.

Further, the sample processing cavity is provided with a sample inlet, and the sample inlet is not provided with a cover; the lysate or the eluent is placed in a reagent bottle, and the reagent bottle mouth and the sample inlet can be matched in a sealing way.

The sample inlet of the nucleic acid detecting device provided by the invention can be intercepted by the nucleic acid adsorption film even if dust enters the sample inlet without arranging a cover.

After nucleic acid isothermal amplification, the content of the nucleic acid to be detected is greatly increased, and the risk of biological pollution exists, so that when the eluent is added by adopting the reagent bottle, the bottle mouth of the reagent bottle can be directly screwed with the sample inlet, and the sealing and pollution preventing effects are achieved.

In another aspect, the present invention provides a detection apparatus, comprising:

a chamber for processing a sample; a cavity for sample reaction in which reaction products of the sample are obtained; and a detection chamber for detecting a reaction product;

the processing sample cavity has a first position, a second position, and a third position; the sample reaction cavity is provided with a first position and a second position;

when the processing sample cavity and the sample reaction cavity are in the first position, the processing sample cavity, the sample reaction cavity and the detection cavity are not in fluid communication with each other.

In some aspects, when the process sample cavity is in the second position, the process sample cavity is in fluid communication with the sample reaction cavity, and the sample reaction cavity is not in fluid communication with the detection cavity.

In some aspects, the sample reaction chamber is in fluid communication with the detection chamber when the process sample chamber is in the third position.

In some aspects, the sample reaction chamber is in the first position when the process sample chamber is moved from the first position to the second position; the sample reaction chamber is positioned at the second position or the sample reaction chamber is moved from the first position to the second position when the processing sample chamber is moved from the second position to the third position.

In some aspects, the sample reaction chamber is in fluid communication with the detection chamber when the sample reaction chamber is in the second position.

In some aspects, the sample processing chamber is moved from the second position to the third position simultaneously with the movement of the sample reaction chamber from the first position to the second position, or the sample processing chamber is moved simultaneously with the movement of the sample reaction chamber, thereby driving the movement of the sample processing chamber from the second position to the third position and driving the movement of the sample reaction chamber from the first position to the second position.

In some modes, the cavity for processing the sample, the cavity for reacting the sample and the detection cavity are sequentially arranged from top to bottom.

In some embodiments, the process sample chamber, the sample reaction chamber, and the like are rotated to effect a transition from one position to another.

In some aspects, the rotation comprises two rotations; the first rotation causes the sample processing cavity to move from a first position to a second position, and the sample reaction cavity and the detection cavity remain stationary; after the first rotation is completed, the process sample chamber is in fluid communication with the sample reaction chamber, and the sample reaction chamber is not in fluid communication with the detection chamber.

In some embodiments, the first rotation is a single rotation of the process sample chamber, with the sample reaction chamber and detection chamber stationary.

In some aspects, the second rotation moves the process sample chamber from the second position to the third position while the sample reaction chamber moves from the first position to the second position; after the second rotation is completed, the processing sample cavity, the sample reaction cavity and the detection cavity are in fluid communication.

In some embodiments, the second rotation is a rotation of the process sample chamber and the sample reaction chamber together, and the detection chamber remains stationary.

In some aspects, the process sample cavity is used to extract nucleic acid species from a sample.

In some embodiments, the sample reaction chamber is used for amplification of nucleic acid species to produce amplification products.

In some aspects, the target test agent is a nucleic acid; the processing sample cavity is used for adsorbing and purifying nucleic acid in the sample; the sample reaction cavity is used for providing a nucleic acid amplification reagent to perform a nucleic acid amplification reaction.

In some embodiments, the detection chamber is used to detect the amount of amplification product or the presence of amplification product.

In some aspects, the sample reaction chamber comprises reagents for nucleic acid amplification, wherein the reagents are present in a dry state.

In some embodiments, the detection chamber includes a lateral flow test strip that is used to detect the amount of amplification product or the presence of amplification product.

In some aspects, the invention provides a nucleic acid detection device, the processing sample cavity for adsorbing nucleic acids in a sample; the sample reaction cavity is used for completing nucleic acid amplification reaction; the detection chamber is used for detecting nucleic acid in the amplified product.

In some modes, after the first rotation, the processing sample cavity is communicated with the sample reaction cavity, and the extracted nucleic acid is transferred to the sample reaction cavity for nucleic acid amplification; after the second rotation, the sample reaction cavity is communicated with the detection cavity, and the amplified product is transferred to a transverse flow test strip in the detection cavity for detection.

In some modes, the processing sample cavity is provided with a sample inlet, a sample inlet channel is connected below the sample inlet, and a nucleic acid adsorption film is fixed at the bottom of the sample inlet channel and is used for adsorbing nucleic acid in a sample.

In some aspects, the sample reaction chamber is provided with a reaction chamber and a filter paper reservoir; the filter paper storage tank is internally provided with filter paper for adsorbing redundant samples; a nucleic acid amplification reaction membrane or a nucleic acid amplification reaction drying reagent is fixed in the reaction cavity; the detection cavity is provided with a sample adding hole, and the reaction product starts to be detected after entering the transverse flow test strip from the sample adding hole.

Further, when the processing sample cavity, the sample reaction cavity and the detection cavity are not communicated, a sample inlet of the processing sample cavity is vertically communicated with the filter paper storage tank; when the processing sample cavity is communicated with the sample reaction cavity, a sample inlet of the processing sample cavity is vertically communicated with the reaction cavity of the sample reaction cavity; when the sample processing cavity, the sample reaction cavity and the detection cavity are all communicated, the sample inlet of the sample processing cavity, the reaction cavity of the sample reaction cavity and the sample adding hole of the detection cavity are all vertically communicated.

The invention completes the amplification and detection of nucleic acid by twice rotation, the nucleic acid is firstly adsorbed by the nucleic acid adsorption film in the processing sample cavity, the nucleic acid adsorption film adsorbed with nucleic acid is transferred to the upper part of the amplification reaction cavity by the first rotation, and the nucleic acid on the nucleic acid adsorption film is washed into the reaction cavity by the eluent in a free falling manner through the shortest distance for isothermal amplification; the reaction cavity and the amplified products are transferred to the transverse flow test strip through the second rotation, the amplified products drop into the test strip in a free falling mode for detection, and the use is more convenient and flexible and no error occurs.

Therefore, the invention ensures that each sample adding or reagent adding can reach the target area in a free falling mode by designing a mode of twice rotation, has compact and small structural design, can improve the efficiency without additional drainage facilities, and improves the detection sensitivity.

In some modes, the processing sample cavity and the sample reaction cavity are cylindrical in shape, and can be conveniently held and rotated.

In some modes, the processing sample cavity comprises an upper cover and a lower cover, a sample inlet is arranged on the upper cover, a sample inlet channel is connected below the sample inlet, and a nucleic acid adsorption film is fixed at the bottom of the sample inlet channel and is used for adsorbing nucleic acid in a sample; the lower cover comprises an outer wall and a bottom surface, and the bottom surface is provided with a through hole; the upper cover and the lower cover are fixed together, and the through hole is always aligned with the sample injection channel.

In some modes, the diameter of the sample inlet is 5-10 mm, the sample inlet channel is cylindrical, the diameter of the bottom surface is consistent with the diameter of the sample inlet, the sample inlet is 15-25 mm high, the sample directly extends downwards from the sample inlet, and the sample reaches the nucleic acid adsorption film through the sample inlet channel.

After the sample enters from the sample inlet and is accelerated by a sample inlet channel with a certain height, the sample can impact the nucleic acid adsorption film with a larger force, so that the nucleic acid adsorption film is promoted to more fully adsorb nucleic acid in the sample, and the nucleic acid detection sensitivity is improved.

In some embodiments, the nucleic acid-adsorbing membrane is a circular membrane, and silica or polysiloxane (silica gel) membrane may be used; the diameter of the sample injection valve is consistent with the diameter of the sample inlet or slightly larger than the diameter of the sample inlet. The nucleic acid adsorption film is fixed at the bottom of the sample injection channel, and the outlet of the bottom of the sample injection channel is required to be completely covered, and after the sample enters from the sample injection port, the sample must completely pass through the nucleic acid adsorption film, and almost all the nucleic acid in the sample is adsorbed by the nucleic acid adsorption film.

In some modes, the periphery of the side wall of the upper cover is provided with a convex block, the lower cover is provided with a groove matched with the convex block in the side wall, the upper cover and the lower cover are respectively fixed together through the convex block and the groove, that is, the positions of the upper cover and the lower cover are fixed, the through hole on the bottom surface of the lower cover is always aligned with the sample injection channel, and only the rotation can be carried out together, but the upper cover or the lower cover cannot be independently rotated.

In some modes, the diameter of the side wall of the upper cover is slightly smaller than that of the side wall of the lower cover, so that the upper cover can be sleeved in the side wall of the lower cover, and after the upper cover and the lower cover are combined, the upper plane of the upper cover is exactly the highest Duan Jiping of the side wall of the lower cover.

In the initial position, namely before rotating, the sample injection channel of the upper cover of the sample cavity is processed, the through hole of the lower cover is positioned above the filter paper storage tank, and the sample injection channel, the through hole and the filter paper storage tank are vertically distributed in a straight line. Thus, at the time of initial sample application, the nucleic acid in the sample is adsorbed by the nucleic acid adsorption membrane, and the remaining liquid enters the filter paper storage tank through the nucleic acid adsorption membrane and is adsorbed by the filter paper in the tank.

The reaction cavity is used for nucleic acid amplification reaction, and in order to make the detection process more convenient and flexible, the invention needs to place fixed and dry nucleic acid amplification reaction reagent in the reaction cavity in advance, thereby avoiding the need of adding a plurality of reagents from a sample adding port for many times in the detection process, and preventing the reagent from being adsorbed by a nucleic acid adsorption film of the sample adding port to influence the isothermal amplification reaction and leading to detection errors.

Further, the outer wall of handling the sample cavity is equipped with first rotatory buckle, and the lateral wall of sample reaction cavity is equipped with the first rotatory draw-in groove of fretwork, and when first rotatory, first rotatory buckle removes the other end from the one end of first rotatory draw-in groove, and first rotatory buckle and first rotatory draw-in groove quantity are more than 1.

In some modes, the diameter of the lower end of the side wall of the lower cover of the processing sample cavity is reduced, so that the side wall of the lower cover can be sleeved into the side wall of the sample reaction cavity, and the processing sample cavity and the sample reaction cavity are combined together to form a regular cylinder, so that the appearance is attractive and convenient to rotate.

In some modes, more than 1 raised first rotary buckles, such as 2 first rotary buckles, are arranged on the periphery of the lower end of the side wall of the lower cover of the sample processing cavity, and are symmetrically distributed at the lower end of the outer wall; the upper part of the outer wall of the sample reaction cavity is provided with more than 1 first rotary clamping grooves, such as 2 first rotary clamping grooves, which are symmetrically distributed on the upper part of the outer wall; the first rotary clamping groove is a strip-shaped transverse hollow groove arranged on the outer wall, and when the lower end of the side wall of the lower cover is sleeved into the side wall of the sample reaction cavity, the protruding first rotary clamping buckle is just positioned in the hollow first rotary clamping groove.

In some modes, the left end and the right end of the first rotary clamping groove are respectively provided with a convex limiting block. When the rotary clamping device is at the initial position, the first rotary clamping buckle is positioned at the leftmost end of the first rotary clamping groove and is blocked and fixed by the limiting block; after the nucleic acid extraction is finished (after the sample is added and the nucleic acid in the sample is adsorbed by the nucleic acid adsorption film), the first rotation buckle starts to rotate for the first time, the first rotation buckle needs to overcome the blocking of the left end limiting block, slides rightwards along the first rotation clamping groove until the first rotation buckle overcomes the right end limiting block to enter the rightmost end, is blocked and fixed by the limiting block, cannot rotate rightwards any more, and at the moment, the sample injection channel and the nucleic acid adsorption film are just transferred to the upper part of the amplification reaction cavity, so that the nucleic acid flushing and isothermal amplification reaction are prepared.

The first rotation is to fix the nucleic acid detecting device by one hand and rotate the nucleic acid detecting device by the other hand, and is to treat the rotation of the sample cavity relative to the sample reaction cavity.

The nucleic acid adsorption membrane is provided with a first position and a second position, wherein the first position is that the nucleic acid adsorption membrane is positioned above the filter paper storage tank, and the second position is that the nucleic acid adsorption membrane is positioned above the reaction cavity; when the first rotary buckle is positioned at one end of the first rotary clamping groove, the nucleic acid adsorption film is positioned at the first position, and when the first rotary buckle moves to the other end of the first rotary clamping groove, the nucleic acid adsorption film is positioned at the second position.

When the nucleic acid adsorption film is positioned at the first position, the nucleic acid detection device is positioned at the initial position, and sample addition can be started; after one rotation is completed, the nucleic acid adsorption film is positioned at a second position, and the sample introduction channel, the through hole and the reaction cavity are vertically distributed in a straight line at the moment, so that the eluent can be added and the nucleic acid isothermal amplification reaction can be started.

It will be appreciated that the first and second positions of the nucleic acid adsorbing membrane are relative to the sample reaction chamber, and that the nucleic acid adsorbing membrane and the sample reaction chamber move together when the second rotation is performed, so that there is no change in the position of the nucleic acid adsorbing membrane relative to the sample reaction chamber, but that the nucleic acid adsorbing membrane also has a third position relative to the detection chamber, because after the second rotation the nucleic acid adsorbing membrane and the reaction chamber are together transferred over the loading well of the lateral flow strip.

In some embodiments, the detection chamber includes a test strip upper cover and a test strip lower cover, and the lateral flow test strip is disposed in the test strip upper cover and the test strip lower cover for detecting nucleic acid in the amplified product.

The test strip upper cover is equipped with application of sample hole and testing result observation window, and application of sample hole aligns the application of sample testing position of test strip, and testing result observation window aligns the testing result reading window of test strip for read testing result.

Further, a hollowed second rotary clamping groove is formed in the side wall of the detection cavity; the outer wall of the sample reaction cavity is provided with a second rotary buckle, when the second rotary buckle rotates for the second time, the second rotary buckle moves from one end of the second rotary clamping groove to the other end, and the number of the second rotary buckle and the number of the second rotary clamping groove are more than 1.

In some modes, the upper cover of the test strip is provided with a cylindrical groove, and the groove wall is provided with a second rotary clamping groove; the lower part of the outer wall of the sample reaction cavity is provided with a second rotary buckle, and when the sample reaction cavity rotates relative to the detection cavity, the second rotary buckle moves from one end of the second rotary clamping groove to the other end.

In some modes, the diameter of the lower part of the outer wall of the sample reaction cavity is reduced, so that the lower part of the outer wall of the sample reaction cavity can be sleeved into the cylindrical groove of the upper cover of the test strip, and the sample reaction cavity and the detection cavity are combined together to form a regular cylinder from the sample processing cavity to the sample reaction cavity and then to the cylindrical groove.

In some modes, more than 1 raised second rotary buckles, such as 2 second rotary buckles, are arranged at the lower part of the outer wall of the sample reaction cavity, and are symmetrically distributed at the lower end of the outer wall; the upper part of the groove wall of the cylindrical groove is provided with more than 1 second rotary clamping grooves, such as 2 second rotary clamping grooves, which are symmetrically distributed on the groove wall; the second rotary clamping groove is a strip-shaped transverse hollowed-out groove arranged on the groove wall, and when the lower part of the outer wall of the sample reaction cavity is sleeved into the cylindrical groove, the convex second rotary clamping buckle is just positioned in the hollowed-out second rotary clamping groove.

In some modes, the left end and the right end of the second rotary clamping groove are respectively provided with a convex limiting block. In the first rotation process, the positions of the second rotary buckle and the second rotary clamping groove are kept unchanged all the time, and the second rotary buckle is positioned at the leftmost end of the second rotary clamping groove and is blocked and fixed by a limiting block at the left end; after the nucleic acid amplification reaction is completed, the second rotation is started, the second rotation buckle needs to overcome the blocking of the left end limiting block, slides rightwards along the second rotation clamping groove until the second rotation buckle overcomes the right end limiting block to enter the rightmost end and is blocked and fixed by the limiting block, and at the moment, the nucleic acid adsorption film and the reaction cavity are jointly transferred to the upper part of the sample adding hole of the detection reagent strip.

The second rotation is to fix the nucleic acid detecting apparatus by one hand and rotate the nucleic acid detecting apparatus by the other hand in the middle or upper part of the nucleic acid detecting apparatus, and is the rotation of the sample reaction chamber relative to the detecting chamber.

In some aspects, the processing sample chamber rotates with the sample reaction chamber as the sample reaction chamber rotates relative to the detection chamber, and the nucleic acid-adsorbing membrane is positioned over the reaction chamber.

Further, the number of the first rotary clamping buckles, the first rotary clamping grooves, the second rotary clamping buckles and the second rotary clamping grooves is 2, and the first rotary clamping buckles, the first rotary clamping grooves, the second rotary clamping buckles and the second rotary clamping grooves are symmetrically distributed.

Further, the reaction cavity is a cylindrical cavity body and is communicated up and down; the periphery of the lower end of the cylindrical cavity is provided with a gasket, and when the reaction cavity rotationally slides on the bottom surface of the cylindrical groove, liquid in the reaction cavity cannot leak out of the reaction cavity under the protection of the gasket; the bottom surface of cylinder type groove is equipped with the application of sample hole, and when the reaction chamber rotated to the application of sample hole top, the amplification product drops into the test paper strip by the application of sample hole with the mode of free fall and carries out nucleic acid detection.

The reaction chamber is a cylinder with a vertically through structure, and has no upper bottom surface or lower bottom surface. The absence of the upper bottom surface facilitates the direct washing of the sample from the upper nucleic acid-adsorbing membrane into the reaction chamber; the absence of the lower bottom surface is for facilitating the movement of the amplification reaction product in the reaction chamber to the sample-loading hole of the bottom plate inside the nucleic acid detecting device, and at this time, the liquid in the reaction chamber can be smoothly dropped onto the nucleic acid detecting reagent strip due to the absence of the bottom plate support.

The reaction chamber does not have the lower bottom surface, but the reaction chamber is arranged on the test strip lower cover to the reaction chamber lower extreme periphery is equipped with the packing ring, makes the liquid in the reaction chamber can not leak in the gap of reaction chamber lateral wall and test strip lower cover surface, and when accomplishing the amplification reaction and carrying out the rotation for the second time, the reaction chamber will slide at the test strip lower cover surface, has had the protection of packing ring, even slides also can not make the reaction liquid leak outside the reaction chamber.

In some embodiments, a gasket groove is provided at the periphery of the lower end of the reaction chamber for fixing the gasket position.

Further, a layer of drainage groove is arranged on the inner wall of the reaction cavity; the drainage groove is formed by equidistantly arranging a plurality of trapezoid columns arranged on the inner wall; a drainage canal is formed between two adjacent trapezoid columns.

Since the sample used for nucleic acid amplification is very small, it is a very critical step to ensure the sensitivity of nucleic acid detection how to ensure that the sample is completely guided to the reaction chamber. Through all setting up the drainage groove around the reaction chamber inner wall, can be to the sample drainage to the reaction intracavity that awaits measuring to the maximum, avoid omitting.

In some embodiments, the trapezoidal column extends from the upper end of the reaction chamber to the lower end.

The trapezoidal column can drain the sample from the upper end of the reaction cavity to the lower end all the time from the upper end of the reaction cavity to the lower end, mainly because the immobilized reaction film or the dried reagent for the nucleic acid amplification reaction is arranged at the lower end of the reaction cavity, the sample can be fully contacted with the immobilized reaction film or the dried reagent only by draining the sample from the upper end of the reaction cavity to the lower end, and thus the amplification reaction efficiency is improved.

In some modes, the upper end of the trapezoid column is provided with an upward narrowing bench; the width of the drainage channel is 0.1-0.5 mm.

The upper end of the trapezoid column is narrowed upwards, and the trapezoid table can be contacted with the nucleic acid adsorption film above, so that the sample from above can be absorbed to enter the drainage channel for full drainage.

In some embodiments, the ramp height is 0.2mm, narrowing upward.

The width of the drainage channels formed between each trapezoidal column is consistent, the narrower the width of the drainage channels is, the better the drainage effect is, but the difficulty in the manufacturing process is increased due to the fact that the drainage channels are too narrow, and therefore the proper width of the drainage channels is required to be selected.

In some modes, the number of the trapezoid columns is 13, and the number of the drainage channels is 13, and the drainage channels are uniformly distributed along the inner wall of the reaction cavity.

In some embodiments, the width of the drainage channel is 0.5mm.

In some modes, the width of the bottom of the trapezoid column is 0.9mm, the protruding distance from the inner wall of the reaction cavity to the outside is 1.25mm, and the height is 3.7mm.

The arrangement of the width of the drainage channels and the number of the drainage channels (the number of the drainage channels determines the number and arrangement of the trapezoid columns, so that the bottom width of the trapezoid columns is determined), and whether the drainage effect can be improved to the greatest extent is determined. According to the invention, the diameter of the cross section of the cylinder of the reaction cavity is 6mm, and a great number of researches prove that when the width of the drainage channel is 0.5mm, the width of the bottom of the trapezoid column is 0.9mm, and the outward protruding distance from the inner wall of the reaction cavity is 1.25mm, a better drainage effect can be achieved.

Further, a heating hole is arranged at the bottom of the detection cavity and used for providing a heat source for the nucleic acid amplification reaction of the sample reaction cavity.

In some embodiments, the heating hole is located on the lower cover of the test strip, and the heating hole is located right below the reaction chamber and is used for providing a heat source for nucleic acid amplification reaction.

In some modes, a heating hole is arranged for the heat source of the nucleic acid isothermal amplification reaction, and the heating hole is matched with isothermal heating equipment, and the isothermal heating equipment provides a heat source for the reaction cavity through the heating hole so as to perform the isothermal amplification reaction.

In some embodiments, the heat source for the isothermal nucleic acid amplification reaction may also be disposed directly inside the nucleic acid detecting apparatus, thus eliminating the need for additional isothermal heating devices.

It can be appreciated that the reaction chamber has a first position and a second position, wherein the first position is that the reaction chamber is positioned above the heating hole, and the second position is that the reaction chamber is positioned above the sample adding hole; when the second rotary buckle is positioned at one end of the second rotary clamping groove, the reaction cavity is positioned at the first position, and when the second rotary buckle moves to the other end of the second rotary clamping groove, the reaction cavity is positioned at the second position.

The reaction chamber is in the first position unchanged from when the nucleic acid detecting means is in the initial position until after the first rotation is completed, because the first rotation is just a rotation of the sample processing chamber, and the sample reaction chamber has not been moved.

When the second rotation is carried out, the nucleic acid adsorption film and the sample reaction cavity rotate together, so that the reaction cavity is rotated from the upper part of the heating hole to the upper part of the sample adding hole, and the sample adding detection is carried out.

Further, the nucleic acid amplification reaction membrane includes a first reaction membrane and a second reaction membrane; the first reaction film is tiled at the lower end of the reaction cavity, and the second reaction film and the first reaction film are horizontally arranged, or the second reaction film is vertically arranged above the first reaction film, or the second reaction film and the first reaction film are vertically arranged.

In some embodiments, the first reaction membrane is an immobilized reaction membrane of a recombinase reagent required for a nucleic acid amplification reaction; the second reaction membrane is an immobilized reaction membrane of a PEG buffer reagent required by nucleic acid amplification reaction; studies prove that the recombinase reagent and the PEG buffer reagent must be separately placed before the amplification reaction, otherwise, the recombinase is easily wrapped by PEG in the amplification reaction, thereby influencing the amplification reaction efficiency, and therefore, two layers of immobilized reagent reaction membranes are required to be arranged.

Further, the first reaction membrane is tiled at the lower end of the reaction cavity, and the second reaction membrane is positioned at the center of the cylinder of the reaction cavity and is vertically arranged above the first reaction membrane.

Experiments prove that the placement positions of the two layers of reaction films can also influence the drainage effect of the sample, and when the second reaction film is vertically placed above the first reaction film and is positioned at the middle position, the drainage effect can be improved, so that the amplification reaction efficiency is improved, and the nucleic acid detection sensitivity is improved.

Further, two nucleic acid amplification reaction dry reagents, namely a first dry reagent and a second dry reagent, are fixed in the reaction cavity; the first dry reagent and the second dry reagent are separated by a parting bead arranged on the bottom surface of the reaction cavity.

The first dry reagent is a recombinase reagent required by nucleic acid amplification reaction; the second dry reagent is a PEG buffer reagent required by nucleic acid amplification reaction; firstly, respectively arranging the two sides of the parting bead, and drying or freeze-drying to obtain a dry reagent; during detection, the two reagents are mixed after being redissolved by adding the eluent to start the nucleic acid amplification reaction, so that the parting bead cannot be too high, and the two reagents are difficult to mix after being redissolved.

In yet another aspect, the present invention provides a method for nucleic acid detection using a nucleic acid detection system, comprising the steps of:

(1) Adding a lysate into the sample to complete sample pyrolysis;

(2) Adding the cracked sample from the sample adding port;

(3) Completing the first rotation;

(4) Adding the eluent from the sample adding port;

(5) Starting a constant temperature heating device to amplify nucleic acid;

(6) Finishing the second rotation;

(7) The detection results are read from the observation window of the upper cover of the reagent strip.

The nucleic acid detection system provided by the invention has the characteristics of simplicity, convenience, integration and the like, reduces the requirements of routine nucleic acid detection on laboratories, equipment and personnel skills, and can be used in sites, small clinics, pet stores and the like.

In some embodiments, the nucleic acid detection systems provided herein can be used for nucleic acid detection of pathogens.

In some embodiments, the nucleic acid detection systems provided herein can be used for nucleic acid detection of pet pathogens (e.g., feline herpesvirus), large livestock pathogens, plant pathogens, or food pathogens.

The beneficial effects of the invention are as follows:

1. PEG in the nucleic acid amplification reagent and the recombinase are separately dried and fixed, and are mixed when the reaction is carried out, so that the nucleic acid detection sensitivity is remarkably improved, and missed detection and false detection are prevented;

2. the formulas of the lysate and the eluent are improved, so that the influence on the nucleic acid amplification reaction can be prevented, the detection sensitivity can be improved, and the operation process is simplified;

3. The structure of the nucleic acid detection device is improved, the nucleic acid detection can be completed through a twice rotation mode, the sample addition or the reagent addition at each time can reach a target area in a free falling mode, the efficiency can be improved without additional drainage facilities, and the detection sensitivity is improved;

4. the method is simple, small and exquisite, low in cost and simple and convenient to operate;

5. the detection sensitivity is high;

6. the detection time is fast;

7. can be used for people, animals, etc., and has wide application range.

Drawings

FIG. 1 is a schematic diagram showing the structure of a nucleic acid detecting apparatus in example 1;

FIG. 2 is a sectional view of the nucleic acid detecting apparatus in example 1;

FIG. 3 is a schematic diagram showing the structural separation of the nucleic acid detecting apparatus in example 1;

FIG. 4 is a schematic view showing the structure of the upper cover of the sample processing chamber in example 1;

FIG. 5 is a schematic view showing the structure of the lower cover of the sample processing chamber in example 1;

FIG. 6 is a schematic view showing the structure of a sample processing chamber in example 1;

FIG. 7 is a cross-sectional view of the processing sample chamber in example 1;

FIG. 8 is a schematic structural view of a sample reaction chamber in example 1;

FIG. 9 is a schematic diagram showing the structural separation of the detection chamber in example 1;

FIG. 10 is a schematic diagram of the structure of a lateral flow test strip in example 1;

FIG. 11 is a schematic view of the reaction chamber structure (from bottom to top) of the sample reaction chamber in example 1;

FIG. 12 is a schematic view showing the separation of the reaction chamber structure (from bottom to top) of the sample reaction chamber in example 1;

FIG. 13 is a schematic view of the reaction chamber structure (from top to bottom) of the sample reaction chamber in example 1, in which a first reaction membrane and a second reaction membrane are disposed, the second reaction membrane being disposed vertically above the first reaction membrane;

FIG. 14 is a schematic view of the reaction chamber structure (from top to bottom) of the sample reaction chamber in example 1, wherein a spacer is disposed in the middle of the reaction chamber;

FIG. 15 is a diagram showing the rotation and the respective state changes of the nucleic acid detecting apparatus in example 2;

FIG. 16 is a schematic diagram showing the structure of a nucleic acid detecting apparatus according to example 3 after sealing in combination with a reagent bottle;

FIG. 17 is a standard colorimetric card of the test results of the lateral flow test strip in example 4.

Detailed Description

The structures and terms of art to which the present invention pertains are further described below, as understood and interpreted in accordance with the general terms of art unless otherwise specified.

Detection of

Detection indicates the assay or testing for the presence of a substance or material, such as, but not limited to, a chemical substance, an organic compound, an inorganic compound, a metabolic product, a drug or drug metabolite, an organic tissue or a metabolite of an organic tissue, a nucleic acid, a protein or a polymer. In addition, the detection indicates the amount of the test substance or material. Further, assays also refer to immunoassays, chemical assays, enzymatic assays, and the like.

Sample of

The sample or sample that the detection device of the present invention can detect or the collector can collect includes biological fluids (e.g., case fluids or clinical samples). Liquid or fluid samples may be derived from solid or semi-solid samples, including fecal matter, biological tissue, and food samples. The solid or semi-solid sample may be converted to a liquid sample using any suitable method, such as mixing, mashing, macerating, incubating, dissolving, or digesting the solid sample with enzymatic digestion in a suitable solution (e.g., water, phosphate solution, or other buffer solution). "biological samples" include animal, plant and food samples, including, for example, urine, saliva, blood and components thereof, spinal fluid, vaginal secretions, sperm, feces, sweat, secretions, tissues, organs, tumors, cultures of tissues and organs, cell cultures and media derived from humans or animals. Preferably the biological sample is urine, preferably the biological sample is saliva, sputum, nasal secretions and the like. Food samples include food processed materials, end products, meat, cheese, wine, milk and drinking water. Plant samples include plants, plant tissues, plant cell cultures and media derived from any plant. An "environmental sample" is derived from the environment (e.g., a liquid sample from a lake or other body of water, a sewage sample, an earth sample, groundwater, seawater, and a waste liquid sample). The environmental sample may also include sewage or other wastewater.

Any analyte may be detected using a suitable detection element or test element of the present invention. The nucleic acid in blood, saliva, urine is preferably detected by the present invention. With the treatment sample chamber 2 of the present invention, any of the above forms of sample, whether initially solid or liquid, can be collected, provided that such liquid or liquid sample is capable of being adsorbed by the nucleic acid adsorbing element in the treatment sample chamber 2, the nucleic acid adsorbing element being located within the treatment sample chamber 2 and capable of adsorbing nucleic acid in the liquid sample or fluid sample, such that the nucleic acid in the fluid sample is retained in the adsorbing element. The nucleic acid adsorbing element may be any material capable of adsorbing nucleic acids in a liquid, such as sponge, filter paper, polyester fiber, gel, nonwoven fabric, cotton, polyester film, yarn, flocking, etc. The present invention preferably employs the nucleic acid-adsorbing membrane 9 as a nucleic acid-adsorbing element.

Downstream and upstream

Downstream or upstream is divided with respect to the direction of liquid flow, typically liquid or fluid flows from upstream to downstream regions. The downstream region receives liquid from the upstream region and liquid may also flow along the upstream region to the downstream region. Here, the flow direction of the liquid is generally divided, for example, by capillary force, so that the liquid can flow against gravity in a direction opposite to the gravity, and the flow direction of the liquid is divided into upstream and downstream. For example, as shown in FIG. 10, the nucleic acid detecting apparatus 1 of the present invention has a sample inlet 7 for applying a sample, a sample processing chamber 2, a sample reaction chamber 3 and a detection chamber 4, wherein the sample is applied from the sample inlet 7 and then enters the sample processing chamber 2, the sample processing chamber 2 adsorbs nucleic acid in the sample by a nucleic acid adsorption film 9, the nucleic acid adsorption film 9 adsorbed with the nucleic acid is transferred to the upper part of an amplification reaction chamber 15 of the sample reaction chamber 3 by a first rotation, and the nucleic acid on the nucleic acid adsorption film 9 is washed by a shortest distance in a free falling manner by an eluent to the inside of the reaction chamber 15 for isothermal amplification; the processing sample cavity 2 is positioned at the upstream of the sample reaction cavity 3, and the sample reaction cavity 3 is positioned at the downstream; the reaction chamber 15 of the sample reaction chamber 3 and the amplified product are transferred to the transverse flow test strip 25 of the detection chamber 4 through the second rotation, the amplified product is dripped into the test strip 25 in a free falling mode for detection, the use is more convenient and flexible, errors can be avoided, the sample reaction chamber 3 is positioned at the upstream of the detection chamber 4, and the detection chamber 4 is positioned at the downstream. According to the invention, through a mode of designing twice rotation, each sample adding or reagent adding can be ensured to reach a downstream target area from an upstream to a downstream in a free falling mode, and the structure design is compact and small, so that the efficiency can be improved without additional drainage facilities, and the detection sensitivity is improved.

Fluid communication

Fluid communication refers to the ability of a liquid to flow from one location to another, where the flow may be directed through some physical structure. By physical structures is generally meant that liquid passes over the surfaces of the physical structures, or spaces within the structures, and flows, either passively or actively, to another location, the passive being generally induced by external forces, such as capillary flow, air pressure, etc. The flow can be the liquid because of the self action (gravity or pressure) or the passive flow, the fluid with the air pressure action can be the homeotropic flow or the reverse flow, or the fluid can be forced to flow from one position to the other position under the air pressure action. Communication herein does not necessarily mean that a liquid is necessarily present, but merely in some cases indicates a connection or state between two objects, and if a liquid is present, it may flow from one object to another. Here, it means a state where two objects are connected, in contrast, if there is no liquid communication state between the two objects, if there is liquid in or on one object, the liquid cannot flow into or on the other object, and such a state is a non-communication, non-liquid communication state.

TestingElement

By "test element" is meant herein that the element that can detect whether a sample or specimen contains an analyte of interest can be referred to as a test element, and such detection can be based on any of the principles of technology, immunology, chemistry, electronics, optics, molecular, nucleic acid, physics, etc. The test element may be a lateral flow test strip that detects multiple analytes. Of course, other suitable test elements may be employed in the present invention.

Various test elements may be combined together for use in the present invention. One form is a test strip or a lateral flow test strip. Test strips for analyzing analytes (e.g., nucleic acids, etc.) in a sample may be in various forms, such as immunoassay or chemical analysis. The test strip can adopt an analysis mode of a non-competition method or a competition method. The test strip generally comprises a bibulous material having a sample application area, a reagent area and a test area. The fluid or liquid sample is applied to the sample application region and flows by capillary action to the reagent region. In the reagent zone, the sample binds to the reagent if the analyte is present. The sample then continues to flow to the detection zone. Other reagents, such as molecules that specifically bind to the analyte, are immobilized in the detection zone. These reagents react with and bind the analyte (if present) in the sample to the region, or to a reagent in the reagent region. The label for displaying the detection signal is present in a separate label zone from the reagent zone.

A typical non-competitive assay format is one in which a signal is generated if the sample contains an analyte and no signal is generated if the sample does not contain an analyte. In competition methods, a signal is generated if the analyte is not present in the sample, and no signal is generated if the analyte is present.

The test element can be a test paper, and can be made of a material which absorbs or does not absorb water. The test strip may comprise a variety of materials for liquid sample transfer. One of the test strips may be coated with another material, such as a filter paper, on a nitrocellulose membrane. One region of the test strip may be of one or more materials and another region of the test strip of a different material or materials. The test strip may be adhered to a support or hard surface for improving the strength of the pinch test strip.

The analyte is detected by the signal generating system, e.g., using one or more enzymes that specifically react with the analyte, and the composition of the one or more signal generating systems is immobilized on the analyte detection zone of the test strip using the method of immobilizing a specific binding material on the test strip as described previously. The signal generating substance may be on the sample application zone, reagent zone, or test zone, or the entire test strip, and the substance may be impregnated with one or more materials of the test strip. The solution containing the signal is applied to the surface of the test strip or one or more materials of the test strip are immersed in the solution containing the signal. The test paper added with the signal-containing substance solution is dried.

The various zones of the test strip may be arranged in the following manner: the sample adding zone, the reagent zone, the detection zone, the control zone, the liquid sample absorbing zone and the liquid sample absorbing zone. The control zone is located behind the detection zone. All zones may be arranged on a strip of paper of only one material. Different materials may be used for the different regions. Each zone may be in direct contact with the liquid sample or the different zones may be arranged in accordance with the direction of flow of the liquid sample, with the ends of each zone being connected to and overlapping the front end of the other zone. The material used may be a material with good water absorption such as filter paper, glass fiber or nitrocellulose membrane. The test strip may take other forms.

A commonly used reagent strip is a nitrocellulose membrane reagent strip, i.e. the detection area comprises a nitrocellulose membrane (NC) on which specific binding molecules are immobilized to display the detection result; but also cellulose acetate film or nylon film, etc. Such as reagent strips or devices containing reagent strips as described in some of the following patents: US 4857453; US 5073484; US 5119831; US 5185127; US 5275785; US 5416000; US 5504013; US 5602040; US 5622871; US 5654162; US 5656503; US 5686315; US 5766961; US 5770460; US 5916815; US 5976895; US 6248598; US 6140136; US 6187269; US 6187598; US 6228660; US 6235241; US 6306642; US 6352862; US 6372515; US 6379620; and US 6403383. The test strips disclosed in the above patent documents and similar devices with test strips can be used in the test element or test device of the present invention for the detection of an analyte, for example in a sample.

The test strips used in the present invention may be so-called lateral flow test strips (Lateral flow test strip), the specific construction and detection principles of which are well known to those of ordinary skill in the art. A typical test strip comprises a sample collection area or application area 51, a label area 52, a detection area 53 and a bibulous area 54, the sample collection area comprising a sample receiving pad, the label area comprising a label pad, the bibulous area comprising a bibulous pad, wherein the detection area comprises a necessary chemical, such as an immunological or enzymatic chemical, capable of detecting the presence of an analyte. A commonly used test strip is a nitrocellulose membrane strip, i.e., the test area 52 comprises a nitrocellulose membrane on which specific binding molecules are immobilized to reveal the resultant area of the test; but also can be a cellulose acetate film or a nylon film, etc.; of course, a detection result control area may be further included downstream of the detection area, and typically, the control area and the detection area are formed in a horizontal line, which is the detection line 55 or the control line 56. Such test strips are conventional, although other types of strips that utilize capillary action for testing are possible. In addition, the test strip typically carries a dry reagent component, such as an immobilized antibody or other reagent, which, upon encountering the liquid, flows along the strip with capillary action, and with the flow, dissolves the dry reagent component in the liquid, thereby allowing the dry reagent in the zone to react to the next zone for the necessary test. The liquid flow is mainly by capillary action. May be used in the detection device of the present invention, or may be disposed in the detection chamber in contact with the liquid sample, or may be used to detect the presence or amount of analyte in the liquid sample entering the detection chamber.

In addition to the above-described test strips or the lateral flow test strips themselves are used to contact a liquid sample to test the liquid sample for the presence of an analyte. The test element according to the application can itself be used as a detection device for detecting an analyte in a sample, so that the detection device itself is here equivalent to the test element. For example, after the fluid sample is mixed with the treatment fluid, the fluid sample is directly tested by the test element. As will be described in greater detail below, the test element may be used alone to detect when the receiving means is described as processing a fluid sample.

Nucleic acid

The analyte of the present application is a nucleic acid.

The term "nucleic acid" includes any compound and/or substance that can be incorporated into an oligonucleotide strand. Exemplary nucleic acids for use in accordance with the present application include, but are not limited to, DNA, RNA including messenger RNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and the like, as described in detail herein.

The term "deoxyribonucleic acid", "DNA" or "DNA molecule" refers to a molecule consisting of two strands (polynucleotides), each strand comprising monomeric unit nucleotides. Nucleotides are linked to each other in the strand by covalent bonds between the sugar of one nucleotide and the phosphate of the next nucleotide, creating an alternating sugar-phosphate backbone. The nitrogenous bases of two separate polynucleotide strands are hydrogen bonded together to produce double-stranded DNA.

The term "ribonucleic acid", "RNA" or "RNA molecule" refers to a strand of at least 2 base-glycosyl-phosphate combinations. In one embodiment, the term includes compounds consisting of nucleotides, wherein the sugar moiety is ribose. In another embodiment, the termini include RNAs and RNA derivatives in which the backbone is modified. In one embodiment, the RNA may be in the form of tRNA (transfer RNA), snRNA (microRNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, small inhibitory RNA (siRNA), microRNA (miRNA), and ribozymes. The use of siRNA and miRNA has been described (Caudy A et al, genes & development 16:2491-96 Andrefferences cittherein). In addition, these forms of RNA may be single stranded, double stranded, triple stranded or quadruplex stranded. In another embodiment, the term also includes other types of artificial nucleic acids that have backbones but the same bases. In another embodiment, the artificial nucleic acid is PNA (peptide nucleic acid). PNAs contain peptide backbones and nucleotide bases and are capable of binding to DNA and RNA molecules in another embodiment. In another embodiment, the nucleotide is a modified oxetane. In another embodiment, the nucleotide is modified by replacing one or more phosphodiester linkages with phosphorothioate linkages. In another embodiment, the modified nucleic acid comprises any other variant of the phosphate backbone of a natural nucleic acid known in the art. Those of ordinary skill in the art are familiar with the use of phosphorothioate nucleic acids and PNAs, which describe, for example, neilsen P E, currOpin Struct Biol 9:353-57; and Raz N Ket al BiochemBiophys Res Commun.297:1075-84. The production and use of nucleic acids is well known to those skilled in the art and the description thereof, molecular Cloning, (2001), sambrook and Russell, eds. And Methods in Enzymology: methods for molecular cloningin eukaryotic cells (2003) Purchio and G.C.fa each represents a separate embodiment of the invention.

As used herein, the term "nucleic acid" includes one or more of the following types: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polynucleotides (containing D-ribose) and any other type of polynucleotide, which are N-glycosides of purine or pyrimidine bases or modified purine or pyrimidine bases, including abasic sites. The term "nucleic acid", as used herein, also includes polymers of ribonucleosides or deoxyribonucleosides covalently bonded, typically through phosphodiester linkages between subunits, but in some cases through phosphorothioates, methylphosphonates, and the like. "nucleic acid" includes single-and double-stranded DNA and single-and double-stranded RNA. Exemplary nucleic acids include, but are not limited to, gDNA; hnRNA; mRNA; rRNA, tRNA, microrna (miRNA), small interfering RNA (siRNA), micronucleolar RNA (snoRNA), micronuclear RNA (snRNA), and microtemporal RNA (stRNA), and the like, and any combination thereof.

Modified nucleotides

In some embodiments, the mRNA comprises modified nucleotides, wherein the modified nucleotides are selected from one or more of the following: 2-amino adenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyl adenosine, 5-methyl cytidine, C-5 propynyl-uridine, 2-amino adenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methyl cytidine, 2-amino adenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxo-adenosine, 8-oxo-guanosine, O (6) -methyl guanine, pseudouridine, N-1-methyl-pseudouridine, 2-thiouridine and 2-thiocytidine; methylated base; inserting a base; 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose; phosphorothioate groups and 5' -N-phosphoramidite linkages. Modified nucleotides as described in PCT/CN2020/074825, PCT/CN 2020/106696.

Detection device

The detection device is used for detecting whether an analyte is contained in a sample, and the nucleic acid detection device provided by the invention sequentially comprises a sample processing cavity 2, a sample reaction cavity 3 and a detection cavity 4 from top to bottom. The processing sample chamber 2 refers to receiving a sample of a detection device to perform mixing or processing of the sample, such as adsorption, elution, and processing of a liquid or liquid sample by the nucleic acid adsorption film 9. The processing sample chamber 2 is not specifically present for receiving the detection means, and may be present alone, having the function of processing a fluid sample alone. The processing sample cavity 2 is used for extracting nucleic acid in a sample, and when the processing sample cavity 2 rotates relative to the sample reaction cavity 3, the nucleic acid can be transferred to the sample reaction cavity 3 for nucleic acid amplification; the sample reaction cavity 3 is used for completing nucleic acid amplification reaction, and when the sample reaction cavity 3 rotates relative to the detection cavity 4, amplified products can be transferred to the detection cavity 4; the detection chamber 4 is used for detecting nucleic acids in amplified products.

Two rotations

Because the nucleic acid detection steps are more, and the detection device needs to be as exquisite and simple as possible, the change of the mutual position relation of the processing sample cavity 2, the sample reaction cavity 3 and the detection cavity 4 needs to be exquisite in design along with the detection flow, so that the nucleic acid extraction, amplification and detection can be completed by mutual matching in a limited space, the functions of sample adding, nucleic acid purification, isothermal amplification and immunochromatography reading detection result are integrated on one device, a simple and small nucleic acid detection device is prepared, the nucleic acid detection can be completed through a twice rotation mode, the sample adding or reagent adding each time can reach a target area in a free falling mode, and additional drainage facilities are not needed.

The invention provides a processing sample cavity 2 in a detection device 1 for extracting nucleic acid substances from a sample, and a sample reaction cavity 3 for amplifying the nucleic acid substances to generate amplified products. The detection chamber 4 is used to detect the amount of amplified product or whether amplified product is present.

The sample processing chamber 2 has a first position 201, a second position 202 and a third position 203 (FIG. 15), and the nucleic acid adsorbing membrane 9 inside the sample processing chamber 2 also has a first position 901, a second position 902 and a third position 903; sample reaction chamber 3 has first position 301 and second position 302, while reaction chamber 15 in sample reaction chamber 3 also has first position 1501 and second position 1502.

When the process sample chamber 2 and the sample reaction chamber 3 are in the first position 201, the process sample chamber 2, the sample reaction chamber 3 and the detection chamber 4 are not in fluid communication with each other.

When the process sample chamber 2 is in the second position 202, the process sample chamber 2 is in fluid communication with the sample reaction chamber 3, and the sample reaction chamber 3 is not in fluid communication with the detection chamber 4.

When the process sample chamber 2 is in the third position 203, the sample reaction chamber 3 is in fluid communication with the detection chamber 4.

When the processing sample cavity 2 is moved from the first position 201 to the second position 202, the sample reaction cavity 3 is in the first position 301; when the process sample chamber 2 is moved from the second position 202 to the third position 203, the sample reaction chamber 3 is located at the second position 302, or the sample reaction chamber 3 is moved from the first position 301 to the second position 302.

When sample reaction chamber 3 is in second position 302, sample reaction chamber 3 is in fluid communication with detection chamber 4.

While the processing sample cavity 2 moves from the second position 202 to the third position 203, the sample reaction cavity 3 moves from the first position 301 to the second position 302, or the processing sample cavity 2 moves simultaneously with the sample reaction cavity 3, thereby driving the processing sample cavity 2 to move from the second position 202 to the third position 203 and driving the sample reaction cavity 3 to move from the first position 301 to the second position 302.

The cavity 2 for processing the sample, the cavity 3 for reacting the sample and the detection cavity 4 are sequentially arranged from top to bottom, and the whole appearance is cylindrical. The processing sample cavity 2 and the sample reaction cavity 3 are respectively rotated to realize the conversion of different positions. The rotation includes two rotations; the first rotation moves the process sample chamber 2 from the first position 201 to the second position 202, the sample reaction chamber 3 and the detection chamber 4 remain stationary; after the first rotation is completed, the process sample chamber 2 is in fluid communication with the sample reaction chamber 3, while the sample reaction chamber 3 is not in fluid communication with the detection chamber 4. The first rotation is thus a rotation of the process sample chamber 2 alone, the sample reaction chamber 3 and the detection chamber 4 being stationary. The second rotation moves the process sample chamber 2 from the second position 202 to the third position 203, while the sample reaction chamber 3 moves from the first position 301 to the second position 302; after the second rotation is completed, the processing sample chamber 2, the sample reaction chamber 3 and the detection chamber 4 are in fluid communication. Thus, the second rotation is the joint rotation of the process sample chamber 2 and the sample reaction chamber 3, and the detection chamber 4 remains stationary.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto. The reagents not specifically mentioned in this example are all known products and are obtained by purchasing commercially available products.

Example 1 nucleic acid detecting apparatus according to the present invention

The nucleic acid detecting apparatus according to the present embodiment is shown in FIGS. 1 to 14.

As shown in fig. 1 to 3, the nucleic acid detecting apparatus 1 according to the present embodiment includes, from top to bottom, a sample processing chamber 2, a sample reaction chamber 3, and a detection chamber 4. The processing sample cavity 2 is used for extracting nucleic acid in a sample, and when the processing sample cavity 2 rotates relative to the sample reaction cavity 3, the nucleic acid can be transferred to the sample reaction cavity 3 for nucleic acid amplification; the sample reaction cavity 3 is used for completing nucleic acid amplification reaction, and when the sample reaction cavity 3 rotates relative to the detection cavity 4, amplified products can be transferred to the detection cavity 4; the detection chamber 4 is used for detecting nucleic acids in amplified products.

As shown in fig. 4 and 5, the sample cavity 2 is used for extracting nucleic acid in a sample, and comprises an upper cover 5 and a lower cover 6, wherein a sample inlet 7 is arranged on the upper cover 5, a sample inlet channel 8 is connected below the sample inlet 7, a nucleic acid adsorption film 9 is fixed at the bottom of the sample inlet channel 8, and the nucleic acid adsorption film 9 is used for adsorbing nucleic acid in the sample; the lower cover 6 comprises an outer wall 10 and a bottom surface 11, and the bottom surface 11 is provided with a through hole 12; the upper cover 5 and the lower cover 6 are fixed together, and the through hole 12 is always aligned with the sample introduction channel 8.

Preferably, as shown in fig. 4 to 7, the periphery of the side wall of the upper cover 5 is provided with a protruding block 13, the lower cover 6 is provided with a groove 14 matched with the protruding block 13 through the side wall, the upper cover 5 and the lower cover 6 are respectively fixed together through the protruding block 13 and the groove 14, that is, the positions of the upper cover 5 and the lower cover 6 are fixed, the through hole 12 on the bottom surface of the lower cover 6 is always aligned with the sample injection channel 8, and the upper cover 5 or the lower cover 6 cannot be independently rotated when the upper cover and the lower cover are rotated together.

The diameter of the side wall of the upper cover 5 is slightly smaller than that of the side wall of the lower cover 6, so that the upper cover 5 can be sleeved in the side wall of the lower cover 6, and after the upper cover 5 is combined with the lower cover 6 by arranging the convex ring 20 in the side wall of the lower cover 6, the upper cover upper plane 39 is just flush with the highest section 38 of the side wall of the lower cover.

Preferably, the diameter of the sample inlet 7 is 8mm, the sample inlet channel 8 is cylindrical, the bottom surface diameter is consistent with the sample inlet 7, the height is 19mm, the sample directly extends downwards from the sample inlet 7, and the sample reaches the nucleic acid adsorption membrane 9 from the sample inlet 7 through the sample inlet channel 8. The nucleic acid adsorbing membrane 9 is a circular thin film, and may be a silica or polysiloxane (silica gel) membrane; the diameter of which is identical to or slightly larger than the diameter of the sample inlet 7. The nucleic acid adsorption film 9 is fixed at the bottom of the sample injection channel 8 and is required to completely cover the outlet at the bottom of the sample injection channel 8; after the sample enters from the sample inlet 7 and is accelerated by the sample inlet channel 8 with a certain height, the sample can impact the nucleic acid adsorption film 9 with a larger force, so that the nucleic acid adsorption film 9 is promoted to more fully adsorb nucleic acid in the sample, and the nucleic acid detection sensitivity is improved. Therefore, after the sample enters from the inlet 7, all the nucleic acid passes through the nucleic acid adsorbing membrane 9, and almost all the nucleic acid in the sample is adsorbed by the nucleic acid adsorbing membrane 9.

As shown in fig. 8, a reaction chamber 15 and a filter paper storage tank 16 are arranged at the bottom of the sample reaction chamber 3; the filter paper storage tank 16 is filled with filter paper for adsorbing redundant samples; the reaction chamber 15 is internally provided with a nucleic acid amplification reaction membrane or is fixed with a nucleic acid amplification reaction drying reagent, so that the detection process is more convenient and flexible, multiple reagents need to be added from the sample adding port 7 in the detection process for multiple times, and the detection errors are prevented from being caused by the influence of the adsorption of the reagents by the nucleic acid adsorption membrane 9 of the sample adding port 7 on the isothermal amplification reaction.

As shown in fig. 9, the outer wall 10 of the lower cover 6 of the processing sample cavity 2 is provided with a first rotary latch 17, the upper part of the side wall of the sample reaction cavity 3 is provided with a first rotary latch 18, when the processing sample cavity 2 rotates relative to the sample reaction cavity 3, the first rotary latch 17 moves from one end to the other end of the first rotary latch 18, and the number of the first rotary latch 17 and the number of the first rotary latch 18 are both more than 1.

Referring to fig. 5, the diameter of the lower end 19 of the side wall of the lower cover 6 is reduced, so that the side wall of the lower cover 6 can be sleeved into the side wall of the sample reaction cavity 3 (fig. 8), and the processed sample cavity 2 and the sample reaction cavity 3 are combined together to form a regular cylinder, so that the appearance is more attractive and more convenient to rotate.

Preferably, the periphery of the lower end 19 of the side wall of the lower cover 6 is provided with 2 raised first rotary buckles 17 which are symmetrically distributed at the lower end 19 of the side wall; the upper part of the outer wall of the sample reaction cavity 3 is provided with 2 first rotary clamping grooves 18 which are symmetrically distributed on the upper part of the outer wall; the first rotary clamping groove 18 is a strip-shaped transverse hollowed-out groove arranged on the outer wall, and when the lower end 19 of the side wall of the lower cover is sleeved into the side wall of the sample reaction cavity 3, the protruding first rotary clamping buckle 17 is just positioned in the hollowed-out first rotary clamping groove 18.

As shown in fig. 8, the left end and the right end of the first rotary clamping groove 18 are respectively provided with a protruding limiting block. When in an initial position, the first rotary buckle 17 is positioned at the leftmost end of the first rotary clamping groove 18 and is blocked and fixed by the limiting block 21; after the nucleic acid extraction is completed (after the sample is added and the nucleic acid in the sample is adsorbed by the nucleic acid adsorption film), the first rotary buckle 17 needs to overcome the blocking of the left end limiting block 21 and slide rightwards along the first rotary clamping groove 18 until the first rotary buckle 17 overcomes the right end limiting block 22 to enter the rightmost end, is blocked and fixed by the limiting block 22, cannot continue to rotate rightwards, and at the moment, the sample introduction channel 8 and the nucleic acid adsorption film 9 are just transferred to the upper part of the amplification reaction cavity 15, so that the nucleic acid flushing and isothermal amplification reaction are prepared.

As shown in FIG. 9, the detection chamber 4 comprises a test strip upper cover 23 and a test strip lower cover 24, and a lateral flow test strip 25 is arranged in the test strip upper cover 23 and the test strip lower cover 24 and is fixed in position for detecting nucleic acid in amplified products. The test strip upper cover 24 is provided with a sample adding hole 32 and a detection result observation window 41, wherein the sample adding hole 32 is aligned with the sample adding detection position of the test strip, and the detection result observation window 41 is aligned with the detection result reading window of the test strip and is used for reading the detection result. The test strip upper cover 23 is provided with a cylindrical groove 26, the sample adding hole 32 is positioned at the bottom surface of the cylindrical groove 26, and the groove wall is provided with a second rotary clamping groove 27; the lower part 28 of the outer wall of the sample reaction chamber 3 is provided with a second rotary catch 29, and when the sample reaction chamber 3 rotates relative to the detection chamber 4, the second rotary catch 29 moves from one end of the second rotary catch 27 to the other end. The number of the second rotary buckles 29 and the second rotary clamping grooves 27 is more than 2. The 2 second rotary buckles 29 are symmetrically distributed at the lower end 28 of the outer wall of the sample reaction cavity 3, and the 2 second rotary clamping grooves 27 are symmetrically distributed on the groove wall of the cylindrical groove 26. The second rotary clamping groove 27 is a strip-shaped transverse hollowed-out groove formed in the groove wall, and when the lower portion 28 of the outer wall of the sample reaction cavity 3 is sleeved into the cylindrical groove 26, the convex second rotary clamping buckle 29 is just positioned in the hollowed-out second rotary clamping groove 27. The left end and the right end of the second rotary clamping groove 27 are respectively provided with a convex limiting block. In the first rotation process, the positions of the second rotary buckle 29 and the second rotary clamping groove 27 are kept unchanged all the time, and the second rotary buckle 29 is positioned at the leftmost end of the second rotary clamping groove 27 and is blocked and fixed by a limiting block 30 at the left end; after the nucleic acid amplification reaction is completed, the second rotation is started, the second rotary buckle 29 needs to overcome the blocking of the left end limiting block 30, slides rightward along the second rotary clamping groove 27 until the second rotary buckle 29 overcomes the right end limiting block 31 to enter the rightmost end and is blocked and fixed by the limiting block 31, and at the moment, the nucleic acid adsorption film 9 and the reaction cavity 15 are jointly transferred to the upper part of the sample adding hole 32 of the detection reagent strip 25.

Preferably, as shown in FIG. 9, the test strip lower cover 24 is provided with a heating hole 33, and the heating hole 33 is located below the reaction chamber 15 for providing a heat source for the nucleic acid amplification reaction. The heat source for the isothermal nucleic acid amplification reaction can be a heating hole 33, the heating hole 33 is matched with a constant temperature heating device, the heating hole can be placed on a circular metal protrusion of any constant temperature heat source, and the constant temperature heating device provides a heat source for the reaction cavity 15 through the heating hole 33 to start and maintain the isothermal amplification reaction. Of course, the heat source for the isothermal nucleic acid amplification reaction may be directly provided inside the nucleic acid detecting apparatus 1, so that it is not necessary to provide a constant temperature heating device additionally.

The structure of the lateral flow test strip 25 is shown in fig. 10, and the lateral flow test strip comprises a sample adding area 51, a marking area 52, a detection area 53 and a water absorbing area 54, wherein the detection area 53 comprises a detection line 55 or a control line 56. In this embodiment, the labeling area 52 is coated with anti-FAM antibody labeled colloidal gold, the detection line 55 is coated with streptavidin, the control line 56 is coated with goat anti-mouse antibody (goat anti-mouse IgG antibody), and the principle of nucleic acid detection is as follows: the amplification primer contains biotin modification, FAM fluorescent groups are modified at the 5' end of the probe, the FAM fluorescent groups are carried at the 5' end of an amplification product after the amplification of the primer probe, the biotin groups are carried at the 3' end of the amplification product, and the target sequence can be smoothly detected through the lateral flow test strip 25.

As shown in FIG. 11, the reaction chamber 15 is a cylindrical chamber, and is vertically penetrated without an upper bottom surface or a lower bottom surface. The absence of the upper bottom surface facilitates the direct washing of the sample from the upper nucleic acid adsorbing membrane 9 into the reaction chamber 15; the absence of the bottom surface is to facilitate movement of the amplification reaction product in the reaction chamber 15 to the sample well 32, and at this time, the liquid in the reaction chamber 15 can be smoothly dropped onto the nucleic acid detecting reagent strip 25 due to the absence of the bottom plate support. A gasket 152 is arranged on the periphery of the lower end 151 of the reaction cavity 15, and when the reaction cavity 15 rotationally slides on the bottom surface of the cylindrical groove 26, the liquid in the reaction cavity 15 cannot leak out of the reaction cavity 15 under the protection of the gasket 152; when the reaction chamber 15 rotates to the position above the sample adding hole 32, the amplified product is dripped into the test strip from the sample adding hole 32 for nucleic acid detection.

The reaction chamber 15 has no lower bottom surface, but the reaction chamber 15 is placed on the test strip lower cover 24, and the gasket 152 can prevent liquid in the reaction chamber 15 from leaking out of the gap between the lower end 151 of the side wall of the reaction chamber and the surface of the test strip lower cover 24, and when the amplification reaction is completed and the amplification reaction is rotated for the second time, the reaction chamber 15 slides on the surface of the test strip lower cover 24, and the reaction liquid cannot leak out of the reaction chamber 15 even if the reaction chamber slides due to the protection of the gasket 152.

Referring to FIG. 12, a gasket groove 153 is provided at the periphery of the lower end 151 of the reaction chamber for fixing the position of the gasket 152.

Referring to FIG. 11, a layer of drainage grooves 154 are formed on the inner wall of the reaction chamber 15; the drainage groove 154 is formed by equidistantly arranging a plurality of trapezoidal columns 155 arranged on the inner wall; a drainage channel 156 is formed between two adjacent trapezoidal columns 155. Since the sample used for nucleic acid amplification is very small, it is a very critical step to ensure the nucleic acid detection sensitivity how to ensure that the sample is completely guided to the reaction chamber 15. By arranging the drainage grooves 154 around the inner wall of the reaction cavity 15, the sample to be measured can be drained into the reaction cavity 15 to the greatest extent, and omission is avoided. Preferably, the trapezoidal column 155 is directly connected from the upper end to the lower end of the reaction chamber 15, so that the sample can be always guided from the upper end to the lower end of the reaction chamber 15, mainly because the immobilized reaction membrane or the dried reagent for the nucleic acid amplification reaction is arranged at the lower end of the reaction chamber 15, and the sample can be fully contacted with the immobilized reaction membrane or the dried reagent only by always guiding the sample from the upper end to the lower end of the reaction chamber 15, thereby improving the amplification reaction efficiency.

Preferably, as shown in fig. 13, the upper end of the trapezoidal column 155 is provided with an upwardly narrowing landing 157; the width of the drainage channel 156 is 0.1-0.5 mm. The upper end of the trapezoidal column 155 is narrowed upward to allow contact with the upper nucleic acid adsorbing membrane 9, thereby helping to absorb the sample from above into the drainage channel 156 for sufficient drainage. In this embodiment, the upwardly narrowing landing 157 is 0.2mm high. The width of the drainage channels 156 formed between each trapezoidal column 155 is consistent, the narrower the width of the drainage channels 156, the better the drainage effect, but the too narrow the drainage channels 156 can make the manufacturing process difficult to upgrade, so that the proper drainage channel width needs to be selected.

Preferably, the number of the trapezoid columns 155 is 13, the number of the drainage channels 156 is 13, the drainage channels are uniformly distributed along the inner wall of the reaction cavity 15, the width of the drainage channels is 0.5mm, the bottom width of the trapezoid columns 155 is 0.9mm, the outward protruding distance from the inner wall of the reaction cavity 15 is 1.25mm, and the height is 3.7mm. The width of the drainage channels 156 and the number of the drainage channels 156 (the number of the drainage channels determines the number and arrangement of the trapezoidal columns, thereby determining the bottom width of the trapezoidal columns), and determining whether the drainage effect can be improved to the greatest extent. In this embodiment, the diameter of the cross section of the cylinder of the reaction chamber 15 is 6mm, and a great amount of researches prove that when the width of the drainage channel 156 is 0.5mm, the bottom width of the trapezoid column 155 is 0.9mm, and the protruding distance from the inner wall of the reaction chamber 15 to the outside is 1.25mm, a better drainage effect can be achieved.

As shown in fig. 13, when the nucleic acid amplification reaction membrane including the first reaction membrane 35 and the second reaction membrane 36 is previously placed in the reaction chamber 15, since the recombinase reagent and the PEG buffer reagent in the amplification reaction reagent must be separately placed before the amplification reaction, otherwise, the recombinase is easily coated with PEG in the amplification reaction, thereby affecting the efficiency of the amplification reaction, it is necessary to provide two immobilized reagent reaction membranes of the first reaction membrane 35 and the second reaction membrane 36. The first reaction film 35 and the second reaction film 36 are arranged in three ways: 1. the first reaction membrane 35 is horizontally laid at the lower end of the reaction cavity 15, and the second reaction membrane 36 and the first reaction membrane 35 are horizontally arranged; 2. the second reaction film 36 is vertically placed above the first reaction film 35; 3. the second reaction membrane 36 is disposed vertically to the first reaction membrane 35. In this embodiment, the 2 nd type is preferable, the first reaction membrane 35 is tiled at the lower end of the reaction chamber 15, the second reaction membrane 36 is located at the center of the cylinder of the reaction chamber 15, and is vertically placed above the first reaction membrane 35. The 2 nd placement mode is preferable because: experiments prove that the placement positions of the two layers of reaction films can also influence the drainage effect of the sample, and when the second reaction film 36 is vertically placed above the first reaction film 35 and is positioned at the middle position, the drainage effect can be improved, so that the amplification reaction efficiency is improved, and the nucleic acid detection sensitivity is improved.

Of course, it is also possible to use a first dry reagent and a second dry reagent, respectively, for immobilizing the nucleic acid amplification reaction dry reagent in the reaction chamber 15; the first dry reagent and the second dry reagent need to be separated by a spacer 37 provided at the bottom of the reaction chamber (FIG. 14). The first dry reagent is a recombinase reagent required by nucleic acid amplification reaction; the second dry reagent is PEG buffer reagent needed by nucleic acid amplification reaction; firstly, respectively placing the two sides of the parting bead 37, and drying or freeze-drying to obtain a dry reagent; in the detection, the nucleic acid amplification reaction is started by adding an eluent to re-dissolve the two reagents and then mixing, so that the spacer 37 cannot be too high, and too high can cause difficulty in mixing after re-dissolution, and in this embodiment, the height of the spacer 37 is 2mm.

Example 2 rotation and State Change Process of nucleic acid detecting apparatus provided by the invention

The nucleic acid detecting apparatus 1 provided in this embodiment can complete amplification and detection of nucleic acid by rotating twice, and the rotation and state change process is shown in fig. 15, wherein (1) in fig. 15 is in an initial state; fig. 15 (2) shows a state after the first rotation; fig. 15 (3) shows a state after the second rotation.

As can be seen from fig. 15, the treatment sample chamber 2 has a first position 201, a second position 202 and a third position 203, and at the same time, the nucleic acid adsorbing membrane 9 inside the treatment sample chamber 2 also has a first position 901, a second position 902 and a third position 903; sample reaction chamber 3 has first position 301 and second position 302, while reaction chamber 15 in sample reaction chamber 3 also has first position 1501 and second position 1502.

Before the nucleic acid detecting apparatus 1 is used (FIG. 15 (1)), the processing sample chamber 2 is in the first position 201 and the nucleic acid adsorbing membrane 9 is also in the first position 901, before the nucleic acid detecting apparatus is used (i.e., before it is rotated; sample reaction chamber 3 is in first position 301 and reaction chamber 15 is also in first position 1501. At this time, the processing sample cavity 2, the sample reaction cavity 3 and the detection cavity 4 are not in fluid communication with each other, that is, the sample introduction channel 8 of the processing sample cavity 2, the reaction cavity 15 of the sample reaction cavity 3 and the sample addition hole 32 of the detection cavity 4 are not in fluid communication yet; however, at this time, the sample introduction channel 8 is located above the filter paper storage tank 16, and the sample introduction channel 8, the through hole 12 and the filter paper storage tank 16 are vertically distributed in a straight line from top to bottom, so that the initial position can start sample introduction, the sample is vertically introduced from the sample introduction port 7, firstly, the sample is contacted with the nucleic acid adsorption membrane 9 after passing through the sample introduction channel 8, the nucleic acid in the sample is adsorbed by the nucleic acid adsorption membrane 9, and the remaining liquid enters the filter paper storage tank 16 through the nucleic acid adsorption membrane 9 and is adsorbed by the filter paper in the filter paper storage tank 16.

After the first rotation (fig. 15 (2)), the treatment sample chamber 2 is rotated from the first position 201 to the second position 202, the nucleic acid adsorbing membrane 9 inside the treatment sample chamber 2 is also rotated from the first position 901 to the second position 902, and the sample reaction chamber 3 is still in the first position 301, and the reaction chamber 15 is still in the first position 1501. At this time, the processing sample cavity 2 is in fluid communication with the sample reaction cavity 3, but the sample reaction cavity 3 is not in fluid communication with the detection cavity 4, that is, the sample introduction channel 8 of the processing sample cavity 2 is turned over the reaction cavity 15 of the sample reaction cavity 3, so that the sample introduction channel 8 is in fluid communication with the reaction cavity 15, and when the reaction cavity 15 does not move, the reaction cavity 15 is not in fluid communication with the sample introduction hole 32 of the detection cavity 4 yet.

The first position 901 of the nucleic acid adsorbing membrane 9 is that the nucleic acid adsorbing membrane 9 is located above the filter paper storage tank 16, and the second position 902 is that the nucleic acid adsorbing membrane 9 is located above the reaction chamber 15; when the first rotary latch 17 is positioned at one end of the first rotary latch 18, the nucleic acid adsorbing membrane 9 is positioned at the first position 901, and when rotated for the first time, the first rotary latch 17 moves to the other end of the first rotary latch 18, and the nucleic acid adsorbing membrane 9 is transferred to the second position 902. After the first rotation, the nucleic acid adsorption film 9 is at the second position 902, and the sample introduction channel 8, the through hole 12 and the reaction cavity 15 are vertically distributed in a straight line, so that the elution solution can be added and the nucleic acid isothermal amplification reaction can be started.

The nucleic acid adsorption film 9 with the adsorbed nucleic acid is transferred to the upper part of the amplification reaction cavity 15 by the first rotation, at the moment, the eluent can be added from the sample inlet 7, passes through the sample inlet channel 8 in a free falling manner, elutes the nucleic acid on the nucleic acid adsorption film 9 to the reaction cavity 15 by the shortest distance, and can be amplified at constant temperature in the reaction cavity 15.

After the second rotation (fig. 15 (3)), the treatment sample chamber 2 is moved from the second position 202 to the third position 203, the nucleic acid adsorbing membrane 9 inside the treatment sample chamber 2 is also rotated from the second position 902 to the third position 903, the sample reaction chamber 3 is moved from the first position 301 to the second position 302, and the reaction chamber 15 is also moved from the first position 1501 to the second position 1502. At this time, the sample reaction cavity 3 is in fluid communication with the detection cavity 4, that is, the reaction cavity 15 of the sample reaction cavity 3 is located above the sample adding hole 32 of the detection cavity 4, and meanwhile, the processing sample cavity 2 is also in fluid communication with the sample reaction cavity 3 and the detection cavity 4, that is, the sample introducing channel 8 of the processing sample cavity 2 is located above the reaction cavity 15, and the sample introducing channel 8, the through hole 12, the reaction cavity 15 and the heating hole 32 are vertically distributed in a straight line.

It will be appreciated that the second rotation is a process of rotating the processing sample chamber 2 and the sample reaction chamber 3 together, and the sample reaction chamber 3 rotates from the first position 301 to the second position 302, and simultaneously, the processing sample chamber 2 is rotated from the second position 202 to the third position 203. The first position 901 and the second position 902 of the nucleic acid-adsorbing membrane 9 are relative to the sample reaction chamber 3, and when the second rotation is performed, the nucleic acid-adsorbing membrane 9 moves together with the sample reaction chamber 3, so that the position of the nucleic acid-adsorbing membrane 9 relative to the sample reaction chamber 3 does not change; that is, when the sample reaction chamber 3 rotates relative to the detection chamber 4, the process sample chamber 2 rotates together with the sample reaction chamber 3, and the nucleic acid adsorbing membrane 9 is always located above the reaction chamber 15. However, the nucleic acid-adsorbing membrane 9 also has a third position 903 with respect to the detection chamber 4 because the nucleic acid-adsorbing membrane 9 and the reaction chamber 15 are transferred together over the well 32 of the detection chamber 4 after the second rotation.

The second rotation causes the reaction chamber 15 to have a first position 1501 where the reaction chamber 15 is located above the heating aperture 33 and a second position 1502 where the reaction chamber 15 is located above the loading aperture 32; when the second rotary latch 29 is located at one end of the second rotary latch 27, the reaction chamber 15 is located at the first position 1501, and when the second rotary latch 29 is moved to the other end of the second rotary latch 27, the reaction chamber 15 is located at the second position 1502.

The first position 1501 of the reaction chamber 15 is unchanged from the time the nucleic acid detecting apparatus 1 is in the initial position until the first rotation is completed, because the first rotation is just a rotation of the processing sample chamber 2 and the sample reaction chamber 3 has not been moved. When the second rotation is performed, the processing sample chamber 2 and the sample reaction chamber 3 are rotated together, and the reaction chamber 15 is rotated from above the heating hole 33 to above the sample addition hole 32, whereby the sample addition detection is performed.

The reaction cavity 15 together with the amplified product is dripped into the test strip 25 through the sample adding hole 32 by the second rotation, and the amplified product is dripped into the test strip 25 in a free falling manner for detection, so that the use is more convenient and flexible and no error occurs. Therefore, through the mode of designing twice rotation, ensure that the application of sample or the reagent of each time can reach the target area with the mode of freely falling, and structural design is compact and small, need not extra drainage facility, just can promote efficiency, improves detection sensitivity.

The nucleic acid detecting apparatus 1 provided in this embodiment can complete the detection of a nucleic acid sample by two simple selection operations, wherein the first rotation is to fix the nucleic acid detecting apparatus 1 by one hand and the second rotation is to rotate at the upper end of the nucleic acid detecting apparatus 1 (e.g., the outer wall of the sample processing chamber 2), which is the rotation of the sample processing chamber 2 relative to the sample processing chamber 3, and the sample processing chamber 3 and the detection chamber 4 are stationary for the rotation of the sample processing chamber 2 alone. The second rotation is to fix the nucleic acid detecting apparatus 1 by one hand, and the other hand is held in the middle part (such as the outer wall of the sample reaction cavity 3) or the upper part of the nucleic acid detecting apparatus 1 to rotate, which is the rotation of the sample reaction cavity 3 relative to the detection cavity 4, so as to treat the rotation of the sample cavity 2 and the sample reaction cavity 3 together, and the detection cavity 4 is kept stationary. The rotation cannot be continued after one time, so that the sequence of the two rotations cannot be wrong, and misoperation cannot be caused.

Example 3 nucleic acid detection System provided by the present invention

The nucleic acid detecting system provided in this embodiment includes the nucleic acid detecting apparatus provided in embodiment 1 and a nucleic acid detecting reagent used in combination therewith. Wherein, the nucleic acid detection reagent comprises a lysate, an eluent and a nucleic acid amplification reaction reagent; as the nucleic acid adsorbing membrane in the nucleic acid detecting apparatus, a polysiloxane silica gel membrane (Shenzhen comma Biotechnology Co., ltd., Y-SM-BC-1) was used.

1. Preparation of lysate

The formula of the lysate comprises the following steps: 0.1M tris (hydroxymethyl) aminomethane, 0.2M ethylenediamine tetraacetic acid, 3M guanidine isothiocyanate, 5% triton 100.

Triton-100 (Triton 100) 5% Tris-HCl 50mmol NaOH 50mmol

2ml of lysate is taken, a reagent bottle is filled in advance, and the size of the reagent bottle mouth is matched with the size of a sample inlet (the sample inlet is not provided with a cover) of the nucleic acid detection device, so that the reagent bottle mouth can be matched in a sealing way.

2. Preparation of nucleic acid amplification reaction reagent

In this example, the nucleic acid amplification reaction reagent is immobilized in advance in the nucleic acid detecting apparatus, and there are mainly two types of (1) an immobilization reaction membrane and (2) a drying reagent.

(1) Immobilization reaction membrane: fiberglass fiber membrane (model 8860), comprising a first reactive membrane and a second reactive membrane, having a size of 6mm diameter and a thickness of 1mm.

Preparation of a first reaction film:

preparing a first reaction solution: 30mM Tris-acetate buffer pH8.0, 50mM potassium acetate, 3mM dithiothreitol, 2mM ATP,20mM creatine phosphate, 100 ng/. Mu.l creatine kinase, 600 ng/. Mu.l E.coli SSB protein, 150 ng/. Mu.l phage uvsX protein, 25 ng/. Mu.l phage uvsY protein, 80 ng/. Mu.l klenow polymerase large fragment (exo-), 50 ng/. Mu.l exonuclease III,200U reverse transcriptase, 450. Mu.M dNTP,420nM each upstream primer, 420nM each downstream primer, 120nM each fluorescent probe.

Taking 0.02ml of the first reaction solution, fixing the first reaction solution on a first reaction film, and drying the first reaction film at 50 ℃.

Preparation of a second reaction film:

preparing a second reaction solution: 5% polyethylene glycol (molecular weight 20000), 0.28M magnesium acetate.

And taking 0.02ml of second reaction solution, fixing the second reaction solution on a second reaction film, and drying the second reaction film at 50 ℃.

(2) And (3) drying the reagent: comprises a first dry reagent and a second dry reagent

The first dry reagent comprises a constituent; the second dry reagent contains a constituent.

A method of prefabricating a first dry reagent and a second dry reagent in a reaction chamber: the reagent is filled in the two sides of the parting bead at the bottom of the reaction cavity, and the nucleic acid detecting device which is pre-filled with the first dry reagent (first reaction liquid) and the second dry reagent (second reaction liquid) is prepared by drying or freeze-drying the reagent.

3. Preparation of eluent

The eluent formula comprises: 0.01M tris (hydroxymethyl) aminomethane, 0.001M ethylenediamine tetraacetic acid, 0.28M magnesium acetate.

Taking 0.06ml of eluent, and pre-filling the eluent into a reagent bottle. The sample inlet of the nucleic acid detection device is not provided with a cover, the reagent bottle mouth and the sample inlet can be matched in a sealing way, when the eluent is added by the reagent bottle, the bottle mouth of the reagent bottle can be directly screwed with the sample inlet and is not taken out (figure 16), the effects of sealing and pollution prevention are achieved in the nucleic acid amplification and detection processes, and biological garbage treatment is carried out after detection, so that biological pollution is prevented.

The embodiment adopts a nucleic acid detection system to detect nucleic acid, wherein a fixed and dried nucleic acid amplification reaction reagent adopts a first reaction membrane and a second reaction membrane, the first reaction membrane is tiled at the lower end of a reaction cavity, and the second reaction membrane is positioned at the central position of the reaction cavity and is vertically arranged above the first reaction membrane. The detection process comprises the following steps:

(1) Adding 2ml of lysate into 0.2ml of sample to complete sample lysis;

(2) Adding 0.5ml of the cracked sample from the sample adding port;

(3) Completing the first rotation of the nucleic acid detecting device;

(4) Adding 0.06ml of eluent from a sample adding port;

(5) Starting a constant temperature heating device to perform nucleic acid constant temperature amplification, and performing temperature and time;

(6) Completing the second rotation of the nucleic acid detecting device;

Example 4 Effect of curing or air drying Process on detection results

In the embodiment, the nucleic acid detection system provided in the embodiment 3 is adopted for detection, the target nucleic acid to be detected is feline herpesvirus FHV-R, the sample to be detected is a 2copies/uL throat swab sample, the amplification condition is 42 ℃ for 12min, and a standard colorimetric card (see figure 17) is used for comparison when the detection result is interpreted.

The primers in this example are:

upstream primer FHV-F: CTATGTTTCTTATGGATATGAGACTTTGTGAT

Downstream primer FHV-R: BIO-ATAGTTTTAACATTTCGACACCATTCATGTAG

Probe FHV-P2:

FAM-CGGTCGCCTTCATATTGGTTGGAACCTTTAAC(THF)AAGTATATGTTCCTAACAG-C3spacer

1. curing process and effect verification

For convenience of preservation and production operations, liquid reagents (first reaction solution and second reaction solution as described in example 3, 0.02 ml) were added to 6mm diameter glass fiber discs with a pipette, respectively, and dried at 37℃under 10% humidity for 2 hours to prepare a solidified reagent. The liquid reagent and the curing reagent are synchronously compared with each other to obtain test results, and the test results are shown in Table 1.

TABLE 1 verification of curing Process Effect

As can be seen from Table 1, the detection results are similar with the curing agent and the liquid agent, there is no obvious difference, and even the average detection sensitivity of the curing agent is slightly higher than that of the common liquid agent.

2. Air drying process and effect verification

The preparation method of the air-drying reagent comprises the following steps: the liquid reagent was added to a specific container without other carriers, dried at 37℃and 10% humidity for 4 hours, and then taken out of the container to be in the form of a sheet. The liquid reagent, the curing reagent and the air-drying reagent are synchronously compared with each other to obtain test results, and the test results are shown in Table 2.

Table 2, verification of air drying Process Effect

As can be seen from Table 2, the detection results are not obviously different by adopting the air-drying reagent, the curing reagent and the liquid reagent, and even the average detection sensitivity of the curing reagent and the air-drying reagent is slightly higher than that of the common liquid reagent.

In summary, the nucleic acid detection system can completely adopt a mode of pre-placing a curing reagent or an air-drying reagent to replace a mode of temporarily adding a liquid reagent, so that the detection process is more convenient.

Example 5 Effect of the solidification mode of PEG and other reagents in nucleic acid amplification reaction reagents on detection results

In order to achieve good amplification effect, PEG is added to the reaction reagent, and the molecular weight of PEG is preferably 20000-40000. The PEG used in this example had a molecular weight of 20000 (Order No. A601790CAS: [25322-68-3], available from Shanghai Co., ltd.). The curing process comprises the following steps: a liquid reagent (0.02 ml) was applied to a glass fiber disk (6 mm diameter, 1mm in thickness) by a pipette, and dried at 37℃under 10% humidity for 2 hours to prepare a solidified reagent. In the process of solidifying the nucleic acid amplification reaction reagent, the following 4 solidification modes are adopted in the embodiment: 1. full mix cure (only one reactive film); 2. the primer probe and enzyme are co-cured (first reaction membrane) and the PEG is individually cured (second reaction membrane); 3. the primer probe and PEG are co-cured (first reaction membrane), and the enzyme is solely cured (second reaction membrane); 4. the enzyme and PEG are co-cured (first reaction membrane) and the primer probe is cured (second reaction membrane) alone. The nucleic acid detection system provided in example 3 was used for detection, the target nucleic acid to be detected was feline herpesvirus FHV-R, the sample to be detected was a 2copies/uL pharyngeal swab sample, the amplification conditions were 42℃for 12min, and a standard colorimetric card (see FIG. 17) was used for comparison when the detection results were interpreted, and the effect of the 4 curing methods on the detection results was compared. The results are shown in Table 3.

TABLE 3 influence of curing modes of PEG and other reagents on detection results

As can be seen from Table 3, the PEG needs to be separated from other reagents during the curing or drying process of the reagents, probably because the PEG can shrink to wrap the enzyme or primer probe during the curing process, and the activity is difficult to recover after the later re-dissolution, so that the isothermal nucleic acid amplification reaction cannot be performed normally. PEG therefore needs to be cured separately.

Example 6 selection of nucleic acid adsorbing Membrane

In this example, the nucleic acid detection system provided in example 3 was used for detection, the nucleic acid adsorption membranes in the nucleic acid detection device were made of different membranes as shown in table 4, the target nucleic acid to be detected was feline herpesvirus FHV-R, the sample to be detected was a 2copies/uL throat swab sample, the amplification conditions were 42 ℃ for 12min, and the detection results were compared using a standard colorimetric card (see fig. 17). Respectively examining the membrane permeation time, the water content, the adsorption capacity and the like of different nucleic acid adsorption membranes, wherein the membrane permeation time is detected by the following method; the detection method of the water content comprises the following steps of; the adsorption capacity detection method comprises the following steps of; the test results are shown in Table 4.

TABLE 4 selection of nucleic acid adsorption membranes

As can be seen from Table 4, the membrane permeation times of different membranes are greatly different, and the sample is directly permeated through the nucleic acid adsorption membrane from the upper side when the nucleic acid detection device provided by the invention is used for detection, and the membrane material with shorter membrane permeation time can rapidly absorb nucleic acid in the sample, so that a GF/C silica membrane or silica gel membrane is preferably used, and the two membranes have small pore diameter, high flow rate, low water content and good adsorption effect, and are particularly suitable for being used as the nucleic acid adsorption membrane, wherein the membrane permeation time of the GF/C silica membrane is shorter, and the adsorption capacity of the silica gel membrane is better (the lower the value of the adsorption capacity is the better).

Example 7 verification of enrichment efficiency of nucleic acid adsorbing Membrane

In this example, a silica gel membrane was used as the nucleic acid adsorption membrane, and the result of detection of nucleic acid adsorbed by the nucleic acid adsorption membrane was examined as the sample size of the membrane was increased, thereby judging the enrichment efficiency of the nucleic acid adsorption membrane. The target nucleic acid to be detected is feline herpesvirus FHV-R, the sample to be detected is a 2copies/uL pharyngeal swab sample, the amplification condition is 42 ℃, the detection method adopts PCR fluorescent detection or product test strip detection respectively, and the result is shown in Table 5.

TABLE 5 verification of enrichment efficiency of nucleic acid adsorption membranes

As can be seen from Table 5, with the increase of the sample volume of the film, both the fluorescence Ct value and the strip depth of the colloidal gold test strip are significantly improved, which indicates that the silica gel film has better nucleic acid enrichment effect.

Example 8 Effect of different lysates on detection results

In the embodiment, the nucleic acid detection system provided in the embodiment 3 is adopted, wherein the lysate is adopted respectively, the influence of different lysates on the nucleic acid detection result is examined, the target nucleic acid to be detected is feline herpesvirus FHV-R, the sample to be detected is a 2copies/uL throat swab sample, the amplification condition is 42 ℃, the amplification condition is 12min, and the detection result is shown in Table 6.

TABLE 6 influence of different lysates on detection results

Sequence number	Formula of lysate	Detection result
			1	Tris+ethylenediamine tetraacetic acid+guanidine isothiocyanate	G3
2	Tris+guanidinium isothiocyanate+Tween 20+NaCl	G4
			3	Tris+guanidinium isothiocyanate+Tween 20	G5
4	Tris+ethylenediamine tetraacetic acid+guanidine isothiocyanate+Tween 20	G6
			5	Tris+ethylenediamine tetraacetic acid+guanidine isothiocyanate+triton 100	G8

As can be seen from Table 6, different lysates have significant influence on the detection result, because some lysates can have adverse effect on isothermal amplification reaction after entering the nucleic acid amplification reaction chamber, but the lysates provided by the invention can not have adverse effect on nucleic acid amplification reaction, so that after nucleic acid in a sample is adsorbed by a nucleic acid adsorption film, the target nucleic acid can be eluted by using eluent without adding a cleaning agent for washing, and then enter the isothermal amplification reaction, and the nucleic acid detection device is particularly suitable for the nucleic acid detection device provided by the invention.

Example 9 Effect of different eluents on detection results

In the embodiment, the nucleic acid detection system provided in the embodiment 3 is adopted, the eluents are respectively adopted, the influence of different eluents on the nucleic acid detection result is examined, the target nucleic acid to be detected is feline herpesvirus FHV-R, the sample to be detected is a 2copies/uL throat swab sample, the amplification condition is 42 ℃, the amplification condition is 12min, and the detection result is shown in Table 7.

TABLE 7 influence of different eluents on the detection results

Sequence number	Eluent formula	Detection result
			1	Tris	Not detected
2	Ethylenediamine tetraacetic acid	Not detected
			3	Magnesium acetate	Not detected
4	Tris+ethylenediamine tetraacetic acid	G5
			5	Tris+ethylenediamine tetraacetic acid+magnesium acetate	G8

As can be seen from table 7, the different eluents have a significant influence on the detection result, because the nucleic acid in the nucleic acid adsorption film is eluted by the eluent, and then enters the reaction chamber to be mixed with the nucleic acid amplification reaction immobilization reagent for isothermal amplification reaction, and the eluent which does not adversely affect the nucleic acid isothermal amplification reaction and even can effectively promote the nucleic acid isothermal amplification reaction needs to be selected, so that the eluent of group 5 is preferably used for the nucleic acid detection device provided by the present invention.

Example 10 Effect of different channel widths and numbers on nucleic acid detection sensitivity

The nucleic acid detection device provided in example 1 is used in this example, and nucleic acid detection is performed in combination with a nucleic acid detection reagent, wherein the width and the number of drainage channels forming the drainage groove are shown in table 8, the nucleic acid detection sensitivity under different setting conditions is detected, the target nucleic acid to be detected is feline herpesvirus FHV-R, the sample to be detected is a 2copies/uL throat swab sample, the amplification condition is 42 ℃ for 12min, and the detection result is shown in table 8.

TABLE 8 influence of different channel widths and numbers on nucleic acid detection sensitivity

As can be seen from Table 8, as the width of the drain channel is reduced, the nucleic acid detection sensitivity is remarkably improved, and the sensitivity improvement is remarkably retarded after the width of the drain channel is reduced to 0.3mm, and it can be seen that the effect of improving the sensitivity by further reducing the width of the drain channel is very limited, and meanwhile, the difficulty in the manufacturing process is remarkably increased and the cost is increased as the width of the drain channel is smaller, so that the width of the drain channel is preferably 0.3-0.7mm, and most preferably 0.5mm.

Meanwhile, the number of the drainage channels has a certain influence on the nucleic acid detection sensitivity, when the width of the drainage channels is not smaller than 0.5mm, the nucleic acid detection sensitivity can be further improved by increasing the number of the drainage channels, but when the width of the drainage channels is smaller than 0.5mm, the effect of increasing the number of the drainage channels on improving the nucleic acid detection sensitivity is not obvious, so that the drainage channels with the width of 0.5mm and the number of the drainage channels of 13 are preferably adopted from the viewpoints of manufacturing difficulty and cost.

Example 11 influence of the height of the sample injection channel on the detection results

The embodiment adopts the nucleic acid detection system provided in embodiment 3, wherein the heights of the sample injection channels in the nucleic acid detection device are respectively selected, and the influence of the heights of different sample injection channels on the detection result is examined. The target nucleic acid to be detected is feline herpesvirus FHV-R, the sample to be detected is a 2copies/uL pharyngeal swab sample, the amplification condition is 42 ℃ for 12min, and the detection result is shown in Table 9.

TABLE 9 influence of sample channel height on detection results

Sequence number	Height of sample injection channel (mm)	Number of detections/number of detections
			1	11	17/20
2	15	19/20
			3	19	20/20
4	23	20/20
			5	27	20/20

As can be seen from table 9, the different sample channel heights directly affect the nucleic acid detection result in the sample, and the reason for this is probably because the different sample channel heights affect the effect of adsorbing the sample by the nucleic acid adsorption film, so that the sample enters from the sample inlet, and after being accelerated by the sample channel with a certain height, the sample can impact the nucleic acid adsorption film with a larger force, thereby promoting the nucleic acid adsorption film to more fully adsorb the nucleic acid in the sample, and improving the nucleic acid detection sensitivity, and therefore, the sample channel with a height of 19mm is preferably used.

Example 12 influence of the arrangement of the first and second reaction films on the detection results

The embodiment adopts the nucleic acid detection system provided in embodiment 3, wherein the nucleic acid amplification reaction reagents adopt a mode of pre-placing the first reaction membrane and the second reaction membrane, and the placing modes of the first reaction membrane and the second reaction membrane are 3 respectively: 1. the second reaction film and the first reaction film are horizontally arranged; 2. the second reaction film is vertically arranged above the first reaction film; 3. the first reaction film and the second reaction film are vertically arranged. The nucleic acid detection sensitivity of the reaction membrane under different placement modes is detected, the target nucleic acid to be detected is feline herpesvirus FHV-R, the sample to be detected is a 2copies/uL throat swab sample, the amplification condition is 42 ℃, the detection result is shown in Table 10 for 12 min.

TABLE 10 influence of different arrangement modes of reaction films on nucleic acid detection sensitivity

Sequence number	Placement mode	Number of detections/number of detections
			1	The first reaction film and the second reaction film are horizontally arranged	17/20
2	The first reaction film is horizontally arranged, and the second reaction film is vertically arranged above the first reaction film	20/20
			3	The first reaction film and the second reaction film are vertically arranged	16/20

It can be seen from table 10 that, by adopting the 2 nd arrangement mode, the first reaction film is horizontally arranged, and the second reaction film is vertically arranged above the first reaction film, so that the nucleic acid detection sensitivity can be obviously improved, and the reason for this is probably that the second reaction film can also play a certain drainage effect, and meanwhile, the mixing effect of reagents in the first reaction film and the second reaction film can also be improved, so that the arrangement mode that the second reaction film is vertically arranged above the first reaction film is preferably adopted.

The nucleic acid detection system provided by the embodiment can be used for detecting on-site, detecting in places such as small clinics, pet stores and the like, and detecting nucleic acid of pathogens; can be used for detecting nucleic acid of pet pathogen, large livestock pathogen, plant pathogen or food pathogen.

The invention is not a matter of the known technology. Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.

Claims

1. A nucleic acid detection system comprising a nucleic acid detection device and reagents, wherein the reagents comprise a lysate, an eluent, and a nucleic acid amplification reaction reagent; the nucleic acid amplification reaction reagent is a reagent after drying and immobilization and comprises a first immobilization reagent and a second immobilization reagent, wherein the first immobilization reagent contains enzyme, and the second immobilization reagent contains PEG.

2. The system of claim 1, wherein the first immobilization reagent further comprises a primer probe, a single-stranded binding protein, a protein cofactor, and DNTP.

3. The system of claim 2, wherein the nucleic acid detecting apparatus comprises a processing sample chamber, a sample reaction chamber, and a detection chamber disposed in this order from top to bottom; the processing sample cavity is used for adsorbing nucleic acid in a sample; the sample reaction cavity is used for completing nucleic acid amplification reaction; the detection cavity is used for detecting nucleic acid in the amplified product; the sample reaction cavity comprises a reaction cavity for performing a nucleic acid amplification reaction; the nucleic acid amplification reaction reagent is placed in the reaction chamber of the sample reaction chamber in advance.

4. The system of claim 3, wherein the nucleic acid amplification reaction reagent is an immobilization reaction membrane or a dry reagent.

5. The system of claim 4, wherein when the nucleic acid amplification reaction reagent is an immobilization reaction membrane, the immobilization reaction membrane comprises a first reaction membrane comprising a first immobilization reagent and a second reaction membrane comprising a second immobilization reagent; when the nucleic acid amplification reaction reagent is a dry reagent, the dry reagent includes a first dry reagent containing a first immobilization reagent and a second dry reagent containing a second immobilization reagent; when the nucleic acid amplification reaction reagent is a dry reagent, a parting bead is required to be arranged at the bottom of the reaction cavity, and the first dry reagent and the second dry reagent are separated by the parting bead.

6. The system of claim 5, wherein the lysate comprises Tris, ethylenediamine tetraacetic acid, guanidine isothiocyanate, and triton 100.

7. The system of claim 6, wherein the process sample chamber comprises a nucleic acid-adsorbing membrane that is a silica GF/C membrane or a silica gel membrane.

8. The system of claim 7, wherein the eluent comprises magnesium acetate, tris and ethylenediamine tetraacetic acid.

9. The system of claim 8, wherein the process sample chamber is provided with a sample inlet, the sample inlet being devoid of a cover; the lysate or the eluent is placed in a reagent bottle, and the reagent bottle mouth and the sample inlet can be matched in a sealing way.