CN118086028A - Integrated nucleic acid detector, nucleic acid detecting apparatus, and nucleic acid detecting method - Google Patents

Abstract

The application relates to the technical field of nucleic acid detection, in particular to an integrated nucleic acid detector, a nucleic acid detection device and a nucleic acid detection method. The integrated nucleic acid detector comprises a column body, a sealing film, a first puncture part and a second puncture part, wherein the column body is provided with a first accommodating channel and a second accommodating channel which penetrate through the column body, the first accommodating channel is used for accommodating a liquid material for detecting nucleic acid, the second accommodating channel is used for accommodating a test strip for detecting nucleic acid, the same end face of the column body is provided with a first opening communicated with the first accommodating channel and a second opening communicated with the second accommodating channel, the column body is connected with the sealing film used for sealing the first opening and the second opening, and the first puncture part and the second puncture part are respectively arranged in the two channels and are in dynamic sealing with the inner wall of the channel and can puncture the sealing film. By adopting the integrated nucleic acid detector, the device or the method, the convenient and rapid individual nucleic acid detection can be performed.

Description

Integrated nucleic acid detector, nucleic acid detecting apparatus, and nucleic acid detecting method

Technical Field

The application relates to the technical field of nucleic acid detection, in particular to an integrated nucleic acid detector, a nucleic acid detection device and a nucleic acid detection method.

Background

The nucleic acid detection is a detection mode for determining whether a human body is infected by viruses by searching whether foreign invasive viruses exist in a biological sample, and is widely applied to detection of respiratory tract virus infection. Since nucleic acid detection generally requires amplification of nucleic acids in a sample to be detected, and is performed under specific laboratory conditions, it is not possible to meet the needs of individuals for diagnosis, and thus, development of an easier, convenient and low-cost personalized nucleic acid detector has become one of the hot spots of current research.

In addition, nucleic acid detection is often used for detection of viral infections, which results in a certain risk of infection for the operator, and therefore, the problem of viral leakage needs to be taken into account in the development of the detector, avoiding the risk of cross-infection.

Disclosure of Invention

Based thereon, one or more embodiments of the present application provide an integrated nucleic acid detector, a nucleic acid detecting apparatus, and a nucleic acid detecting method.

The technical scheme of the application comprises the following contents:

The integrated nucleic acid detector comprises a column body, a sealing film, a first puncture piece and a second puncture piece;

The column body is provided with a first accommodating channel and a second accommodating channel which penetrate through the column body, the first accommodating channel is used for accommodating a material for detecting nucleic acid, the second accommodating channel is used for accommodating a test strip for detecting nucleic acid, the same end face of the column body is provided with a first opening communicated with the first accommodating channel and a second opening communicated with the second accommodating channel, the column body is connected with a sealing film used for sealing the first opening and the second opening, and the column body can be connected with a sample reaction tube in a sealing manner;

the movable first puncture element is arranged in the first accommodating channel and can be in dynamic seal with the inner wall of the first accommodating channel, and the first puncture element can puncture the sealing film;

the movable second puncture member is arranged in the second accommodating channel, the test strip is connected to the second puncture member, the second puncture member can be in dynamic seal with the inner wall of the second accommodating channel, and the second puncture member can puncture the sealing film.

Further, the first puncture element comprises a first sealing part and a first puncture part which are connected, and the first sealing part is dynamically sealed with the inner wall of the first accommodating channel;

the end of the first sealing part far away from the first puncture part protrudes out of the column body to provide a force application site.

Further, the first accommodating channel is a cylindrical channel;

the first sealing part is of a columnar structure matched with the first accommodating channel.

Further, the radial dimension of the first seal is greater than the dimension of the first opening such that the first seal is confined within the first receiving channel.

Further, the second puncture element comprises a second sealing part and a second puncture part which are connected, and the second sealing part is dynamically sealed with the inner wall of the second accommodating channel;

the end of the second sealing part far away from the second puncture part protrudes out of the column body to provide a force application site.

Further, the second accommodating channel is a cylindrical channel;

The second sealing part is of a columnar structure matched with the second accommodating channel.

Further, a part of the test strip is inlaid in the second sealing portion, and the other part of the test strip extends to the second puncture portion.

And one part of the test strip is inlaid in the second sealing part, and the other part of the test strip extends to the second puncture part.

Further, the cylinder is also provided with an air pressure balance channel penetrating through the cylinder, wherein one opening of the air pressure balance channel and the first opening and the second opening are positioned on the same end face of the cylinder;

and a filter element is arranged in the air pressure balancing channel.

Further, the column is further provided with a third accommodating channel penetrating through the column, the third accommodating channel is used for accommodating the diluent for nucleic acid detection, one opening of the third accommodating channel is a third opening, the first opening and the second opening are positioned on the same end face of the column, and the sealing film seals the third opening;

the movable third puncture part is arranged in the third accommodating channel, the third puncture part can be in dynamic seal with the inner wall of the third accommodating channel, and the third puncture part can puncture the sealing film.

Further, the third puncture element comprises a third sealing part and a third puncture part which are connected, and the third sealing part is dynamically sealed with the inner wall of the third accommodating channel;

the end of the third sealing part far away from the third puncture part protrudes out of the column body to provide a force application site.

Further, the third accommodating channel is a cylindrical channel;

The third sealing part is of a columnar structure matched with the third accommodating channel.

Further, the radial dimension of the third seal is greater than the dimension of the third opening such that the third seal is confined within the third receiving channel.

A nucleic acid detecting apparatus comprising:

a nucleic acid extraction tube for extracting nucleic acid to be detected from a biological sample;

A sample reaction tube containing a solid-state amplification reagent for amplifying the nucleic acid to be detected; and

The integrated nucleic acid detector according to any one of the above aspects.

A method for detecting nucleic acid, which adopts the integrated nucleic acid detector according to any one of the above technical schemes, comprising:

obtaining a sample liquid containing nucleic acid to be detected in a sample reaction tube;

And (3) sealing the cylinder of the integrated nucleic acid detector with the sample reaction tube, controlling the first puncture part to puncture the sealing film, enabling the liquid material for nucleic acid detection in the first accommodating channel to enter the sample reaction tube for reaction, controlling the second puncture part to puncture the sealing film after the reaction is finished, enabling the test strip part in the second accommodating channel to enter the sample reaction tube, and reading and analyzing the test strip.

According to the technical scheme, a liquid material and a test strip for nucleic acid detection are respectively pre-stored in a first accommodating channel and a second accommodating channel of an integrated nucleic acid detector, the integrated nucleic acid detector can be in sealing connection with a sample reaction tube containing nucleic acid to be detected, then a first puncture member in the first accommodating channel is controlled to puncture a sealing material at the bottom of the channel and communicate with the sample reaction tube, a liquid reagent for nucleic acid detection enters the sample reaction tube to react, after the reaction is finished, a second puncture member in the second accommodating channel is controlled to puncture the sealing material at the bottom of the channel and enable the test strip to enter the sample reaction tube along with movement of the second puncture member, the liquid in the sample reaction tube wets the test strip, and the nucleic acid detection can be realized by reading and analyzing the test strip. The technical scheme can enable the whole detection process to be carried out under a closed (or relatively closed without nucleic acid leakage) condition, avoids environment pollution caused by aerosol formed by virus-containing samples in the detection process, has higher safety compared with the existing detection device and detection method, has the advantages of simple operation, convenient reading, portability, short time consumption (25-30 min), wider application range, suitability for individual nucleic acid detection and high commercial application value.

Drawings

FIG. 1 is a schematic diagram showing the structure of a cartridge of an integrated nucleic acid detector according to an embodiment of the present application.

FIG. 2 is a schematic diagram showing the structure of a first piercing member of an integrated nucleic acid detector according to an embodiment of the present application.

FIG. 3 is a schematic diagram showing the structure of a second piercing member of an integrated nucleic acid detector according to an embodiment of the present application.

FIG. 4 is a schematic diagram showing the structure of a third piercing member of an integrated nucleic acid detector in accordance with an embodiment of the present application.

FIG. 5 is a schematic diagram showing the structure of an integrated nucleic acid detector according to an embodiment of the present application.

FIG. 6 is a schematic diagram showing a structure of a nucleic acid detecting apparatus according to an embodiment of the present application.

FIG. 7 is a statistical plot of the volume change of each droplet collected from a throat swab sample in example 2 of the present application.

FIG. 8 shows the results of the nucleic acid amplification analysis of the integrated nucleic acid detection method of example 2 of the present application at different reaction temperatures.

FIG. 9 shows the results of analysis of nucleic acid amplification products at 38℃and 36.5℃for different reaction times in example 2 according to the present application.

FIG. 10 shows the results of simultaneous detection of samples containing different common respiratory pathogens at 38deg.C for 20min in example 2 of the present application.

FIG. 11 shows the sensitivity comparison results of InfA/infB integrated nucleic acid detection method and qPCR method and antigen detection method in example 2 of the present application.

Reference numerals illustrate: 1. an integrated nucleic acid detector; 10. a column; 11. a first receiving channel; 111. a fifth opening; 112 a first opening; 12. a second receiving channel; 121. a sixth opening; 122. a second opening; 13. a third receiving channel; 131. a seventh opening; 132. a third opening; 14. an air pressure balancing channel; 141. an eighth opening; 142. a fourth opening; 20. a filter element; 30. sealing film; 41. a first piercing member; 42. a second piercing member; 43. a third piercing member; 2. a nucleic acid extraction tube; 3. sample reaction tube.

Detailed Description

The application is further illustrated below in conjunction with the embodiments and examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it is to be understood that various changes and modifications may be made by one skilled in the art after reading the teachings of the application, and such equivalents are intended to fall within the scope of the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

In the present application, "further," "preferred," etc. are used for descriptive purposes and indicate differences in content but are not to be construed as limiting the scope of the application.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

The recombinant enzyme polymerase amplification technology (RPA, recombinase polymerase amplification), a novel nucleic acid isothermal amplification technology, can realize rapid detection of a target to be detected within 10-30 min at 37-42 ℃; during the reaction of RPA, the recombinant enzyme protein and the primer form a complex, homologous sequence is found in double-stranded DNA, the primer is inserted into the homologous site through the strand displacement activity of the recombinant enzyme, and the single-stranded DNA binding protein stably displaces the DNA strand. The recombinase is then broken down, allowing the 3' strand of the primer to displace and bind to the DNA polymerase, allowing it to elongate the primer, and the target region on the template is amplified exponentially by repeating the process cyclically.

Recombinant enzyme (recombinase) forms a protein-nucleic acid complex with the primer, guides the primer to combine with the template, and plays a role in positioning; helping melting of the DNA template and facilitating strand displacement between the template and the primer.

A single-stranded DNA binding protein (SSB, single stranded DNA binding), which binds to the displaced single-stranded DNA, prevents the DAN pair from binding again.

DNA polymerase (DNA polymerase) synthesizes progeny DNA using dNTPs using DNA as a template.

Endonucleases (endonucleases) that hydrolyze phosphodiester bonds within molecular chains to form oligonucleotides; nfo, endonucleases derived from bacteria and more prone to act on double stranded DNA at the 3' concave end, participate in DNA damage repair.

The probe detection method based on the RPA method is characterized in that a specific probe is added in an RPA system, a tetrahydrofuran modification site is introduced into the probe, the tetrahydrofuran modification nucleotide can realize DNA chain extension with almost 100% efficiency under the action of DNA polymerase, a double-chain luminous group and a quenching group are even at the position close to the tetrahydrofuran modification site, when the probe and a target sequence structure form double-chain DNA, nfo is considered that the DNA is damaged when encountering the tetrahydrofuran modification site in the inspection process, the site on the probe is sheared, thereby releasing a fluorescent group, and the sheared probe has free 3' -OH, so that the probe can be used as a primer to continuously synthesize a new template; multiple PCR assays can be performed using different fluorescent probes.

First aspect

The integrated nucleic acid detector comprises a column body, a sealing film, a first puncture piece and a second puncture piece;

The column body is provided with a first accommodating channel and a second accommodating channel which penetrate through the column body, the first accommodating channel is used for accommodating liquid materials for nucleic acid detection, the second accommodating channel is used for accommodating test strips for nucleic acid detection, the same end face of the column body is provided with a first opening communicated with the first accommodating channel and a second opening communicated with the second accommodating channel, the column body is connected with a sealing film used for sealing the first opening and the second opening, and the column body can be connected with a sample reaction tube in a sealing manner;

A movable first puncture member is arranged in the first accommodating channel, the first puncture member can be in dynamic seal with the inner wall of the first accommodating channel, and the first puncture member can puncture the sealing film;

the second holding passageway is equipped with mobilizable second piercing depth, is connected with the test strip on the second piercing depth, and the second piercing depth can be sealed with the inner wall dynamic of second holding passageway, and the second piercing depth can impale the sealing membrane.

The column body of the integrated nucleic acid detector can be connected with a sample reaction tube in a sealing way, when the first puncture piece punctures the sealing film, the first accommodating channel is communicated with the sample reaction tube, and when the second puncture piece punctures the sealing film, the second accommodating channel is communicated with the sample reaction tube.

In one embodiment, the column is transparent, so that the internal conditions of the first accommodating channel and the second accommodating channel are facilitated, for example, whether the liquid material in the first accommodating channel completely enters the sample reaction tube can be conveniently observed, whether the test strip in the second accommodating channel already enters the sample reaction tube can be observed, and detection is realized.

In one embodiment, the test strip is a chromatographic test strip, and the strip on the test strip can be conveniently read through the column body to analyze the detection result.

Further, the first puncture element comprises a first sealing part and a first puncture part which are connected, and the first sealing part is in dynamic seal with the inner wall of the first accommodating channel;

the end of the first sealing part far away from the first puncture part protrudes out of the cylinder to provide a force application site.

Further, the first accommodating channel is a cylindrical channel;

the first sealing part is of a columnar structure matched with the first accommodating channel.

The columnar structure comprises a cylinder, a square column and the like.

Further, the radial dimension of the first sealing portion is larger than the dimension of the first opening, so that the first sealing portion is limited in the first accommodating channel.

Further, the second puncture element comprises a second sealing part and a second puncture part which are connected, and the second sealing part is dynamically sealed with the inner wall of the second accommodating channel;

the end of the second sealing part far away from the second puncture part protrudes out of the cylinder to provide a force application site.

Further, the second accommodating channel is a cylindrical channel;

the second sealing part is a cylinder matched with the second accommodating channel.

Further, a part of the test strip is inlaid in the second sealing portion, and the other part of the test strip extends to the second puncture portion.

One part of the test strip is inlaid in the second sealing part, and the other part of the test strip extends to the second puncture part.

Further, the cylinder is also provided with an air pressure balance channel penetrating through the cylinder, wherein one opening of the air pressure balance channel, the first opening and the second opening are positioned on the same end face of the cylinder;

the air pressure balancing channel is internally provided with a filter element.

The openings at the two ends of the air pressure balancing channel are not sealed by sealing films, and in one embodiment, after the integrated nucleic acid detector is in sealing connection with the sample reaction tube, the air pressure balancing channel can be used for balancing the air pressure of the sample reaction tube and the outside atmosphere, and liquid in the tube cannot overflow due to pressure change, so that the air pressure balancing channel is safer.

Further, the column is further provided with a third accommodating channel penetrating through the column, the third accommodating channel is used for accommodating the diluent for nucleic acid detection, one opening of the third accommodating channel is a third opening, the first opening and the second opening are positioned on the same end face of the column, and the sealing film seals the third opening;

The third accommodating channel is internally provided with a movable third puncture member, the third puncture member can be in dynamic seal with the inner wall of the third accommodating channel, and the third puncture member can puncture the sealing film.

Further, after the third puncture member punctures the sealing membrane, the third accommodation channel communicates with the sample reaction tube.

Further, the third puncture element comprises a third sealing part and a third puncture part which are connected, and the third sealing part is dynamically sealed with the inner wall of the third accommodating channel;

The end of the third sealing part far away from the third puncture part protrudes out of the column body to provide a force application site.

Further, the third accommodating channel is a cylindrical channel;

The third sealing part is of a columnar structure matched with the third accommodating channel.

Further, the radial dimension of the third sealing portion is greater than the dimension of the third opening, so that the third sealing portion is limited in the third accommodating channel.

Preferably, in the integrated nucleic acid detector, the cartridge has three receiving channels extending through the cartridge: the first accommodating channel, the second accommodating channel and the third accommodating channel are respectively used for accommodating a liquid reagent for nucleic acid amplification, a test strip for nucleic acid detection and a diluent, the same end face of the cylinder is provided with a first opening communicated with the first accommodating channel, a second opening communicated with the second accommodating channel and a third opening communicated with the third accommodating channel, the cylinder is connected with a sealing film used for sealing the first opening, the second opening and the third opening, the cylinder can be in sealing connection with the sample reaction tube, the cylinder is also provided with an air pressure balancing channel penetrating through the cylinder, and one opening of the air pressure balancing channel, the first opening, the second opening and the third opening are positioned on the same end face of the cylinder;

A movable first puncture member is arranged in the first accommodating channel, the first puncture member can be in dynamic seal with the inner wall of the first accommodating channel, and the first puncture member can puncture the sealing film;

a movable second puncture member is arranged in the second accommodating channel, a test strip is connected to the second puncture member, the second puncture member can be dynamically sealed with the inner wall of the second accommodating channel, and the second puncture member can puncture the sealing film;

A movable third puncture element is arranged in the third accommodating channel, the third puncture element can be in dynamic seal with the inner wall of the third accommodating channel, and the third puncture element can puncture the sealing film;

the air pressure balancing channel is internally provided with a filter element.

Second aspect

A nucleic acid detecting apparatus comprising:

A nucleic acid extraction tube for extracting nucleic acid to be detected from the biological sample;

a sample reaction tube containing a solid-state amplification reagent for amplifying a nucleic acid to be detected; and

The integrated nucleic acid detector according to any one of the above aspects.

Third aspect of the invention

A method for detecting nucleic acid, the method adopting the integrated nucleic acid detector of any one of the above technical solutions for detection, comprising:

obtaining a sample liquid containing nucleic acid to be detected in a sample reaction tube;

The column body of the integrated nucleic acid detector is connected with the sample reaction tube in a sealing way, the first puncture part is controlled to puncture the sealing film, so that liquid materials for nucleic acid detection in the first accommodating channel enter the sample reaction tube for reaction, after the reaction is finished, the second puncture part is controlled to puncture the sealing film, and the test strip part in the second accommodating channel enters the sample reaction tube for reading and analyzing the test strip.

Liquid materials for nucleic acid detection include liquid reagents for nucleic acid amplification, e.g.,

Preferably, the nucleic acid detection is performed using an integrated nucleic acid detector having the following technical features:

In the integrated nucleic acid detector, the column has three accommodating channels penetrating the column: the first accommodating channel, the second accommodating channel and the third accommodating channel are respectively provided with a liquid reagent for nucleic acid amplification, a test strip for nucleic acid detection and a diluent, the same end face of the cylinder is provided with a first opening communicated with the first accommodating channel, a second opening communicated with the second accommodating channel and a third opening communicated with the third accommodating channel, the cylinder is connected with a sealing film for sealing the first opening, the second opening and the third opening, the cylinder can be in sealing connection with the sample reaction tube, the cylinder is also provided with an air pressure balancing channel penetrating through the cylinder, and one opening of the air pressure balancing channel, the first opening, the second opening and the third opening are positioned on the same end face of the cylinder;

A movable first puncture member is arranged in the first accommodating channel, the first puncture member can be in dynamic seal with the inner wall of the first accommodating channel, and the first puncture member can puncture the sealing film;

a movable second puncture member is arranged in the second accommodating channel, a test strip is connected to the second puncture member, the second puncture member can be dynamically sealed with the inner wall of the second accommodating channel, and the second puncture member can puncture the sealing film;

A movable third puncture element is arranged in the third accommodating channel, the third puncture element can be in dynamic seal with the inner wall of the third accommodating channel, and the third puncture element can puncture the sealing film;

the air pressure balancing channel is internally provided with a filter element.

The integrated nucleic acid detector with the technical characteristics is used for detecting nucleic acid, and comprises the following steps:

obtaining a sample liquid containing nucleic acid to be detected in a sample reaction tube;

the column body of the integrated nucleic acid detector is connected with the sample reaction tube in a sealing way,

And controlling the first puncture part to puncture the sealing film so that a liquid material for nucleic acid detection in the first accommodating channel enters the sample reaction tube for reaction, controlling the third puncture part to puncture the sealing film after the reaction is finished so that diluent in the third accommodating channel enters the sample reaction tube for dilution, controlling the second puncture part to puncture the sealing film after dilution, and enabling a test strip part in the second accommodating channel to enter the sample reaction tube for reading and analyzing the test strip.

Preferably, the liquid material for nucleic acid detection includes a reaction medium required for an amplification reaction, including a buffer, a buffer salt, and the like; correspondingly, the sample reaction tube contains solid reagents required for the amplification reaction, and these solid reagents are usually lyophilized reagents, including lyophilized reagents for primers, probes, proteins, enzymes, and the like.

Preferably, the diluent is high purity water, such as ddH ₂ O.

Preferably, the test strip for nucleic acid detection is a chromatographic test strip, and after a period of time, the liquid in the sample reaction tube infiltrates the chromatographic test strip, the strip is read to realize nucleic acid detection.

The integrated nucleic acid detector disclosed by the application is used for detecting, compared with the existing detection device and detection method, the integrated nucleic acid detector has higher safety, is simple to operate, convenient to read, portable in device and short in time consumption, and can be used for loading corresponding liquid materials and test strips for detecting nucleic acid into a channel according to different nucleic acid to be detected, wherein the types of the nucleic acid include but are not limited to nucleic acid of influenza A virus, nucleic acid of influenza B virus and pathogen nucleic acid of respiratory diseases; in addition, in the detection method, the liquid material and the solid material for detecting the nucleic acid are respectively arranged in the integrated nucleic acid detector and the sample tube according to the physical and chemical properties of the detection material, so that the detection material does not participate in the reaction when not used, has better storage stability, and can be conveniently mixed when being detected, and the nucleic acid to be detected can be detected efficiently, quickly and conveniently.

By way of example, amplification reagents for amplifying a nucleic acid of a alphavirus include a primer pair having sequences shown in SEQ ID No.1 and SEQ ID No.2, and a probe having a sequence shown in SEQ ID No. 3; the amplification reagent for amplifying the nucleic acid of the B-stream virus comprises a primer pair with the sequences shown as SEQ ID No.4 and SEQ ID No.5 and a probe with the sequences shown as SEQ ID No. 6.

The following are some specific examples.

The experimental parameters not specified in the following specific examples are preferentially referred to the guidelines given in the present document, and may also be referred to the experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer.

The starting materials and reagents referred to in the following specific examples may be obtained commercially or may be prepared by known means by those skilled in the art.

Example 1

Referring to fig. 6, the present embodiment provides a nucleic acid detecting apparatus including: an integrated nucleic acid detector 1, a nucleic acid extraction tube 2 and a sample reaction tube 3.

Referring to fig. 1, 5 and 6, the integrated nucleic acid detector 1 includes a column 10, the column 10 having three receiving channels extending therethrough: the first accommodating channel 11, the second accommodating channel 12 and the third accommodating channel 13 are respectively used for accommodating a liquid reagent for nucleic acid amplification, a test strip for nucleic acid detection and a diluent, the same end face of the cylinder is provided with a first opening 112 communicated with the first accommodating channel 11, a second opening 122 communicated with the second accommodating channel 12 and a third opening 132 communicated with the third accommodating channel 13, the cylinder is connected with a sealing film 30 used for sealing the first opening 112, the second opening 122 and the third opening 132, the cylinder can be in sealing connection with a sample reaction tube, the cylinder is also provided with a gas pressure balancing channel 14 penetrating through the cylinder, and one opening (a fourth opening 142) of the gas pressure balancing channel 14 is positioned on the same end face of the cylinder as the first opening 112, the second opening 122 and the third opening 132.

Referring to fig. 2, a movable first piercing member 41 is provided in the first accommodation channel 11, the first piercing member 41 being capable of being sealed with the inner wall of the first accommodation channel 11, the first piercing member 41 being capable of piercing the sealing film.

Referring to fig. 3, a movable second piercing member 42 is disposed in the second accommodating channel 12, a test strip is connected to the second piercing member 42, the second piercing member 42 can dynamically seal with the inner wall of the second accommodating channel 12, and the second piercing member 42 can pierce the sealing film.

Referring to fig. 4, a movable third piercing member 43 is provided in the third accommodating path, the third piercing member 43 being capable of sealing against the inner wall of the third accommodating path 13, the third piercing member 43 being capable of piercing the sealing film.

Referring to fig. 1, the cylinder is far from the other end face of the sealing film, which has a fifth opening 111 communicating with the first receiving passage 11, a sixth opening 121 communicating with the second receiving passage 12, and a seventh opening 131 communicating with the third receiving passage 13, and one of the openings (eighth opening 141) of the air pressure balancing passage 14 is located at the same end face of the cylinder (the other end face of the cylinder far from the sealing film) as the fifth opening 111, the sixth opening 121, and the seventh opening 132.

Example 2

Samples of 109 suspected influenza A or B virus (Influenza A virus, influenza B virus; infA, infB) infected patients were collected and synchronously detected by control with the nucleic acid detection device of example 1, commercial influenza A and B virus qPCR detection kit and influenza A and B virus and mycoplasma pneumoniae antigen detection kit, respectively, and the clinical diagnostic effect of the established method was analyzed.

Integrated nucleic acid detection reagent for (I) infA and infB

1. In order to establish an infA and infB individuation nucleic acid detection method, respectively comparing and analyzing the published MP gene sequences relatively conserved among the global infA and infB viruses, and selecting a conserved region in the MP gene sequences to carry out primer and probe design based on RPA-nfo (table 1);

Enzyme and protein in the RPA amplification reagent, a primer and a probe together form a freeze-drying reagent in an isothermal amplification reaction tube;

3. The heavy swelling buffer solution, magnesium acetate, diluent (ddH ₂ O) and the chromatographic test strip are integrated into a universal integrated nucleic acid detector.

TABLE 1 RT-RPA-nfo detection primers and probes for influenza A and B viruses

In the sequences shown in Table 1, Y represents C or T, R represents A or G, and THF represents tetrahydrofuran.

(II) sample collection and viral nucleic acid Release

The method integrates sample collection, virus inactivation and nucleic acid release enhancement through a thermostat or boiled water at 95-100 ℃ for 5min (the invention patent authorization number: ZL 202010373017.7), thereby realizing the efficient extraction of virus nucleic acid.

After 10 batches of 600 mu L sample dripping analysis, determining that the first two unstable drops of the volume of the sample after the treatment are firstly discarded, and then one drop of the sample is dripped into an isothermal amplification reaction tube (30.9+/-0.6 mu L) of RT-RPA to ensure the stability of a reaction system, and FIG. 7 is a statistical chart of the volume change of each drop of the sample collection treatment of a throat swab; the data source is mean±sem, mean represents mean, SEM is sampling error of mean, and the sampling accuracy of mean is reflected.

(III) reaction conditions

1. Analysis of reaction temperature

The detection of infA/infB positive samples by using the integrated nucleic acid detection method of infA/infB established by the application is carried out for 30min under the conditions of 36 ℃,36.5 ℃,37 ℃,38 ℃,39 ℃,40 ℃,41 ℃,42 ℃ at different temperatures, the optimal reaction temperature is analyzed, and FIG. 8 shows the result of the nucleic acid amplification analysis of the integrated nucleic acid detection method at different reaction temperatures, wherein 1:36 ℃;2:36.5 ℃;3:37 ℃;4:38 ℃;5:39 ℃;6:40 ℃;7:41 ℃;8:42 ℃; according to the results, the method can achieve better amplification at 36-39 ℃ and has the highest amplification product amount at 38 ℃.

2. Analysis of reaction time

Further analysis the detection of the optimal reaction time at an optimal temperature of 38℃and an extreme field using an underarm temperature of 36.5℃was performed by setting the reaction times for 10min,15min,20min,25min,30min,35min,40min,45min, respectively, and analyzing the changes in the product concentration, FIG. 9 shows the analysis results of nucleic acid amplification products at different reaction times at 38℃and 36.5℃wherein 1: for 10min;2:15min;3:20min;4:25min;5:30min;6:35min;7: for 40min;8:45min. According to the result, the optimal temperature is 38 ℃ and the time reaches 20min, so that the optimal temperature can be obtained; the reaction time is slightly longer at the underarm temperature of 36.5 ℃ and is 25min-30min.

In summary, the nucleic acid detection reaction with integration of infA and infB is performed at 38deg.C for 20min or at an extreme environment of 36.5deg.C for 30min.

(IV) individuation infA and infB nucleic acid detection method specificity and sensitivity

1. Specificity analysis

The method is used for detecting common respiratory pathogens at 38 ℃ for 20min, wherein the common respiratory pathogens comprise 3 strains of 5 strains infA, 5 strains infB, 5 respiratory syncytial viruses and parainfluenza viruses 1,2 and 3, respectively, adenovirus, rhinovirus, metapneumovirus, coronavirus 229E, OC, mycoplasma pneumoniae and chlamydia pneumoniae respectively 2 strains, and simultaneously E.coli TOP10 2 strain and E.coli DH 5a 2 strain, the detection results are shown in figure 10, the detection results are shown as follows in figure 10, the detection results of 5 strains infA and 5 strain infB are all normally detected, the detection results are all negative, the non-specific amplification is avoided, and the interference between infA and infB is avoided.

2. Sensitivity of

The infA and infB positive samples are respectively used for 5-time gradient dilution, the integrated infA and infB double nucleic acid detection method established by the application is used for detection at 38 ℃ for 20min, and commercial influenza A and B virus fluorescent quantitative PCR detection kits and influenza A and B virus and mycoplasma pneumoniae antigen detection kits are used for comparison analysis. As shown in FIG. 11, the nucleic acid detection method integrating the infA and infB is equivalent to the qPCR method in sensitivity but far higher than the antigen detection method in sensitivity (> 125-fold or more, 5-7vs 5-4) according to FIG. 11.

(V) analysis of clinical test results

In order to verify the clinical detection effect of an infA and infB integrated nucleic acid detection method, 109 cases of suspected infA and infB infected patient samples are collected for diagnosis and analysis, and meanwhile, a commercial influenza A and B virus fluorescent quantitative PCR detection kit and a influenza A and B virus and mycoplasma pneumoniae antigen detection kit are used for comparison research.

1. Sample collection, nucleic acid extraction and detection

Each patient simultaneously collected 3 pharyngeal swab samples:

(1) One-to-conventional transfer medium for fluorescent quantitative PCR detection

After the sample is collected, the sample is transported to a detection laboratory by using an ice box;

extracting sample RNA according to the requirement by using an RNA extraction kit;

Taking 5 mu L of the extracted RNA for fluorescent quantitative PCR detection reaction of influenza A virus B virus;

and (3) performing on-machine detection by using an ABI7500 fluorescent quantitative PCR instrument, and judging the result according to the amplification curve.

(2) One-to-antigen sampling tube for antigen detection

Sample collection and cracking;

three drops are dropped into each reaction hole according to the requirement;

The antigen detection results were observed within 15 min.

(3) One portion was used in the RTU collection tube of the present application for infA/infB integrated nucleic acid detection.

The RTU collecting tube collects throat swab samples at the temperature of 95-100 ℃ for 5min;

discarding the first two samples, then dripping one sample into a reaction tube, and connecting the reaction tube to a detector;

the rod was pushed in sequence at 38℃for 20min to carry out the detection reaction and observe the results.

2. Comparison of screening results

And through the comparative detection of 109 patients, judging the standard according to the reagent results. The compliance of the method was 100% compared to qPCR, whereas the antigen detection method was relatively low due to lower sensitivity (table 2).

Table 2.109 clinical sample detection Effect analysis

In summary, the detection method of the application is simple and convenient to operate, does not need expensive equipment, consumes about 25-35 min total time, and is very suitable for detecting respiratory viruses in families, basic medical units and even in extreme environments in the field.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely represent a few embodiments of the present application and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the application should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims (14)

1. The integrated nucleic acid detector is characterized by comprising a column body, a sealing film, a first puncture piece and a second puncture piece;

The column body is provided with a first accommodating channel and a second accommodating channel which penetrate through the column body, the first accommodating channel is used for accommodating liquid materials for nucleic acid detection, the second accommodating channel is used for accommodating a test strip for nucleic acid detection, the same end face of the column body is provided with a first opening communicated with the first accommodating channel and a second opening communicated with the second accommodating channel, the column body is connected with a sealing film used for sealing the first opening and the second opening, and the column body can be connected with a sample reaction tube in a sealing way;

the movable first puncture element is arranged in the first accommodating channel and can be in dynamic seal with the inner wall of the first accommodating channel, and the first puncture element can puncture the sealing film;

the movable second puncture member is arranged in the second accommodating channel, the test strip is connected to the second puncture member, the second puncture member can be in dynamic seal with the inner wall of the second accommodating channel, and the second puncture member can puncture the sealing film.

2. The integrated nucleic acid detector of claim 1, wherein the first piercing member comprises a first sealing portion and a first piercing portion connected, the first sealing portion being in dynamic seal with an inner wall of the first receiving channel;

the end of the first sealing part far away from the first puncture part protrudes out of the column body to provide a force application site.

3. The integrated nucleic acid detector of claim 2, wherein the first receiving channel is a cylindrical channel;

the first sealing part is of a columnar structure matched with the first accommodating channel.

4. The integrated nucleic acid detector of any one of claims 2-3, wherein a radial dimension of the first seal is greater than a dimension of the first opening such that the first seal is confined within the first receiving channel.

5. The integrated nucleic acid detector of claim 1, wherein the second piercing member comprises a second sealing portion and a second piercing portion connected, the second sealing portion being in dynamic seal with an inner wall of the second receiving channel;

the end of the second sealing part far away from the second puncture part protrudes out of the column body to provide a force application site.

6. The integrated nucleic acid detector of claim 5, wherein the second receiving channel is a cylindrical channel;

The second sealing part is of a columnar structure matched with the second accommodating channel.

7. The integrated nucleic acid detector of any one of claims 5 to 6, wherein a portion of the strip is inlaid in the second sealing portion and another portion of the strip extends to the second piercing portion.

8. The integrated nucleic acid detector of any one of claims 1-3, 5-6, wherein the cartridge further has a gas pressure equalization channel extending therethrough, wherein one of the gas pressure equalization channels is located on the same end face of the cartridge as the first opening and the second opening;

and a filter element is arranged in the air pressure balancing channel.

9. The integrated nucleic acid detector according to any one of claims 1 to 3, 5 to 6, wherein the cartridge further has a third accommodation channel penetrating the cartridge, the third accommodation channel being for accommodating a diluent for nucleic acid detection, one of the openings of the third accommodation channel being a third opening located on the same end face of the cartridge as the first opening and the second opening, the sealing film sealing the third opening;

the movable third puncture part is arranged in the third accommodating channel, the third puncture part can be in dynamic seal with the inner wall of the third accommodating channel, and the third puncture part can puncture the sealing film.

10. The integrated nucleic acid detector of claim 9, wherein the third piercing member comprises a third sealing portion and a third piercing portion connected, the third sealing portion being in dynamic seal with an inner wall of the third receiving channel;

the end of the third sealing part far away from the third puncture part protrudes out of the column body to provide a force application site.

11. The integrated nucleic acid detector of claim 10, wherein the third receiving channel is a cylindrical channel;

The third sealing part is of a columnar structure matched with the third accommodating channel.

12. The integrated nucleic acid detector of any one of claims 10-11, wherein a radial dimension of the third seal is greater than a dimension of the third opening such that the third seal is confined within the third receiving channel.

13. A nucleic acid detecting apparatus, comprising:

a nucleic acid extraction tube for extracting nucleic acid to be detected from a biological sample;

A sample reaction tube containing a solid-state amplification reagent for amplifying the nucleic acid to be detected; and

The integrated nucleic acid detector of any one of claims 1 to 12.

14. A nucleic acid detection method, characterized in that it comprises detecting with the integrated nucleic acid detector according to any one of claims 1 to 12, comprising:

obtaining a sample liquid containing nucleic acid to be detected in a sample reaction tube;

And (3) sealing the cylinder of the integrated nucleic acid detector with the sample reaction tube, controlling the first puncture part to puncture the sealing film, enabling the liquid material for nucleic acid detection in the first accommodating channel to enter the sample reaction tube for reaction, controlling the second puncture part to puncture the sealing film after the reaction is finished, enabling the test strip part in the second accommodating channel to enter the sample reaction tube, and reading and analyzing the test strip.