CN114058489A - Anti-pollution nucleic acid amplification product rapid detection device, method and application - Google Patents

Abstract

The invention discloses an anti-pollution nucleic acid amplification product rapid detection device, which comprises a detection inner core and a sealed outer box, wherein a water guide fiber membrane is arranged at a position corresponding to the lower end of a needle head disc; the tube body is used for containing reaction liquid and hybridization liquid, and when the needle head disc punctures the inverted top of the tube body and manually extrudes the side wall of the tube body or shakes the rapid detection device by manual pressing, the reaction liquid passes through the needle head disc under the action of gravity, pressure or centrifugal force and is adsorbed by the water guide fiber membrane to be guided to enter the test strip; the invention also correspondingly discloses a use method of the anti-pollution nucleic acid amplification product rapid detection device. The invention realizes that the liquid in the reaction tube flows out to the designated detection card in a completely closed environment, and has the advantages of good closing effect and prevention of primer aerosol pollution.

Description

Anti-pollution nucleic acid amplification product rapid detection device, method and application

Technical Field

The invention relates to the field of nucleic acid detection, in particular to a device and a method for quickly detecting an anti-pollution nucleic acid amplification product and application.

Background

The novel coronavirus belongs to the genus beta coronavirus, has envelope, and has round or elliptical particle, usually polymorphism, and diameter of 60-140 nm. It is clearly distinguished from SARSr-CoV and MERSR-CoV on the basis of characteristics. The present research shows that the homology with bat SARS-like coronavirus (bat-SL-CoVZC45) reaches more than 85%. Based on current epidemiological investigation, the incubation period is 1-14 days, and is mostly 3-7 days. The clinical manifestations are mostly mild, fever, dry cough and hypodynamia are taken as main manifestations, but severe patients have dyspnea and/or hypoxemia after one week, and severe patients can rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct, coagulation dysfunction, multiple organ failure and the like. Therefore, the early discovery, the early treatment and the early isolation can effectively prevent the spread of diseases and reduce the proportion of severe and critically ill patients.

Nucleic acid extraction has become the most important and basic operation in molecular biology experimental technology, but at present, the extraction scheme of viral nucleic acid is very many and is complicated and simple. The basic population of the population is often the starting point for outbreaks of infectious diseases, and there is a great need to be able to quickly diagnose nucleic acids in situ, which is of great significance in the prevention and control of infectious diseases.

However, according to the detection level and treatment conditions of the field or the basement, a new crown nucleic acid rapid detection product suitable for community and individual field detection is urgently needed to be developed, the detection pressure of medical institutions in severe epidemic areas is relieved, and the cross infection risk of the detected people is reduced.

Disclosure of Invention

In order to solve the problems of the prior art, in one aspect, the present invention provides a rapid nucleic acid amplification product detection device against contamination, comprising an inner detection core and an outer sealing case,

the sealing outer box comprises a sealing cover, an upper shell and a lower shell which are arranged in sequence;

the upper shell comprises an upper end part and a lower end part, the lower end part is buckled with the lower shell to form a first accommodating space, and the detection inner core is arranged in the first accommodating space;

the sealing cover is buckled at the upper end part of the upper shell to form a second accommodating space, an isothermal reaction tube assembly and a needle head disc are arranged in the second accommodating space, and the second accommodating space is used for preventing pollution caused by dropping of the isothermal reaction tube assembly due to device abandonment or mistaken collision of people;

the isothermal reaction tube assembly comprises a tube body and a tube cover which are sequentially connected, and a puncture needle is arranged on the needle head disc; the needle head disc is fixed at a corresponding position below the tube cover, and the puncture needle punctures the tube cover and leads out reaction liquid in the tube body when the isothermal reaction tube assembly moves downwards;

the detection inner core comprises a test strip and a water guide fiber membrane connected with the test strip, and the water guide fiber membrane is arranged at a corresponding position below the needle head disc; when the puncture needle punctures the inverted bottom of the tube body and manually extrudes the side wall of the tube body by downward pressing, the reaction liquid is adsorbed by the water guide fiber membrane through the puncture needle under the action of gravity and pressure and flows into the test strip;

the reaction liquid can be adsorbed and guided by the water-guiding fiber membrane to enter the test strip through the puncture needle under the action of centrifugal force by shaking the sealing device;

and a transparent window is arranged at the upper end part of the upper shell corresponding to the test strip.

As a further improvement of the embodiment of the present invention, the test strip is a colloidal gold test strip or a nucleic acid test strip.

As a further improvement of the embodiment of the present invention, the tube cover includes a circular cover body section and a circumferential wall perpendicularly surrounding the circular cover body section; the inner side of the section of the circular cover body is provided with a circular sealing gasket, and when the puncture needle penetrates through the circular sealing gasket, the pipe orifice of the pipe body is abutted against the circular sealing gasket so as to ensure that liquid cannot flow back and cannot flow down by gravity alone.

As a further improvement of the embodiment of the present invention, an annular sealing ring is further disposed between the tube cover and the tube body, the annular sealing ring is tightly sleeved at the edge of the tube opening of the tube body in the radial direction, and a double-layer sealing structure combining a circular sealing gasket and the annular sealing ring is formed between the tube cover and the tube body, so that the tube cover is prevented from falling off and liquid flows out due to collision of foreign matters during discarding.

As a further improvement of the embodiment of the present invention, three layers of vertically alternate sealing partitions are arranged inside the circumferential wall of the tube cap to be tightly pressed with the annular sealing ring.

As a further improvement of the embodiment of the present invention, the upper housing and the lower housing are constructed in a totally enclosed environment and have a snap-fit member that cannot be opened again once being snapped to prevent the items in the first receiving space from being contaminated or leaking.

As a further improvement of the embodiment of the present invention, the needle disk has a puncturing needle and a circular disk perpendicular to the puncturing needle; the surface of the disc facing the upper shell is provided with an annular bulge, and a guide groove matched with the annular bulge is fixed in the second accommodating space and used for clamping and fixing the needle head disc.

On the other hand, the invention also discloses a rapid detection method of the anti-pollution nucleic acid amplification product, which comprises the following steps:

a) taking down the sealing cover;

b) placing the reaction tube which is well bathed and the hybridization tube which does not need to be bathed into the second containing space of the device for quickly detecting the pollution-proof nucleic acid amplification product of claim 1 respectively and fixedly connecting the reaction tube and the hybridization tube into a guide groove in the upper shell through a needle head disc;

c) pressing the two tube bodies downwards, and puncturing the part with the silica gel cap of the tube cover by the needle head disc;

d) the side wall of the reaction tube and the hybrid liquid tube are manually extruded in sequence, or the sealing device is swung, and liquid in the tube drips through a puncture needle of the needle disc head under the action of gravity, pressure or centrifugal force and is adsorbed and guided by a water guide fiber membrane to enter the test strip;

e) the test paper strip is detected through the transparent window in a visual mode, and the result is judged and read.

As a further improvement of the embodiment of the present invention, the step d) is followed by an operation of covering the sealing cover;

as a further improvement of the embodiment of the present invention, the method further comprises the step of discarding the whole device in a safe place without opening the device after the detection of the step f).

On the other hand, the invention also discloses the application of the anti-pollution nucleic acid amplification product rapid detection device or the anti-pollution nucleic acid amplification product rapid detection method in the aspects of food industry, agriculture, animal husbandry, customs quarantine inspection, gene mutation detection and DNA single nucleotide polymorphism identification.

The invention has the following beneficial effects:

1. the anti-pollution nucleic acid amplification product rapid detection device realizes that liquid in the reaction tube flows out to the specified detection card in a completely closed environment, and has the advantages of good closing effect and prevention of amplification primer aerosol pollution;

2. the device and the method for rapidly detecting the anti-pollution nucleic acid amplification product prevent pollution in the detection process due to good sealing performance, can realize rapid real-time detection according to detection and treatment conditions on site or basic level, and have the advantages of convenient operation, simple use and low specialized requirement;

3. the device and the method for quickly detecting the anti-pollution nucleic acid amplification product have the advantages of being suitable for a new crown nucleic acid quick detection product for community and personal field detection, relieving the detection pressure of medical institutions in areas with serious epidemic situations and reducing the risk of cross infection of examinees.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a longitudinal sectional view of a device for rapid detection of a contamination-preventive nucleic acid amplification product according to an embodiment of the present invention;

FIG. 2 is a schematic structural view of an anti-contamination nucleic acid amplification product rapid detection apparatus according to an embodiment of the present invention in a state where a sealing cap is fastened;

FIG. 3 is a schematic structural view of a device for rapid detection of an anti-contamination nucleic acid amplification product according to an embodiment of the present invention with a sealing cover opened;

the examples in the figures are represented as: 1-detecting an inner core; 11-a test strip; 12-a water-conducting fibre membrane; 2-sealing the outer box; 21-a sealing cover; 22-an upper shell; 221-upper end portion; 222-a lower end portion; 223-a first accommodation space; 224-a second accommodation space; 225-a fixed support; 23-a lower housing; 3-an isothermal reactor tube assembly; 31-a tube body; 32-a tube cover; 33-needle disk; 331-puncturing needle; 332-a disc; 333-guide groove; 4-a circular seal; 5-ring seal ring, 6-transparent window.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The embodiment of the invention provides a device for rapidly detecting an anti-pollution nucleic acid amplification product, which comprises an inner detection core 1 and a sealed outer box 2 as shown in figure 1,

the sealing outer box 2 comprises asealing cover 21, anupper shell 22 and alower shell 23 which are arranged in sequence;

theupper shell 22 comprises anupper end 221 and alower end 222, thelower end 222 is buckled with thelower shell 23 to form a firstaccommodating space 223, and the detection core 1 is arranged in the firstaccommodating space 223;

as shown in fig. 2 and 3, thesealing cover 21 is fastened to theupper end 221 of theupper housing 22 to form a secondaccommodating space 224, afixing bracket 225 may be disposed in the secondaccommodating space 224, and two PCRreaction tube assemblies 3 are clamped on thefixing bracket 225;

in the embodiment of the present invention, the second receivingspace 224 is used to prevent the pollution caused by the dropping of the isothermal reaction tube assembly due to the discarding of the apparatus or the accidental collision of people;

the PCRreaction tube assembly 3 comprises atube body 31, atube cover 32 and aneedle head disc 33 which is communicated with thetube body 31 through thetube cover 32, wherein thetube body 31, thetube cover 32 and the needle head disc are connected in sequence; in the present embodiment,needle hub 33 has alancet 331 and adisk 332 perpendicular to the lancet; thedisk 332 is provided with aguide groove 333 for holding thetube cover 31, and theguide groove 333 is fixedly connected to thefixing bracket 225.

Theneedle disc 33 is fixed at a corresponding position below thetube cover 32, and thepuncture needle 331 punctures thetube cover 3 and leads out the reaction liquid in thetube body 31 when the isothermal reaction tube assembly moves downwards;

the detection inner core 1 comprises atest strip 11 and a waterguide fiber membrane 12 connected with thetest strip 11, wherein part of the waterguide fiber membrane 12 is arranged at a position corresponding to the lower end of theneedle disc 33;

thetube body 31 is used for containing reaction liquid, and when theneedle disc 33 punctures the bottom of thetube body 31 and manually extrudes the side wall of thetube body 31 by pressing downwards, the reaction liquid passes through theneedle disc 33 under the action of gravity and pressure and is adsorbed and guided by the waterguide fiber membrane 12 to enter thetest strip 11;

theupper end 221 of theupper case 22 is provided with a transparent window 6 at a portion corresponding to thetest strip 11.

Wherein, thetest strip 11 is a nucleic acid test strip.

In an embodiment of the present invention, thetube cap 32 includes a circular cap body section and a circumferential wall perpendicularly surrounding the circular cap body section; the inner side of the circular cover body section is provided with acircular sealing gasket 4, and when the puncture needle 311 pierces thecircular sealing gasket 4, the pipe orifice of thepipe body 31 is abutted against thecircular sealing gasket 4 to ensure that the liquid cannot flow back and cannot flow down by gravity alone.

In the embodiment of the present invention, an annular sealing ring 5 is further disposed between thetube cap 32 and thetube body 31, the annular sealing ring 5 is tightly sleeved at the edge of the tube opening of thetube body 31 in the radial direction, and a double-layer sealing structure combining acircular sealing gasket 4 and the annular sealing ring 5 is formed between thetube cap 32 and thetube body 31, so that the tube cap is not dropped and liquid flows out due to collision of foreign matters during discarding.

In other alternative embodiments, three layers of sealing partitions are arranged alternately up and down inside the circumferential wall of thetube cover 32 to tightly press the annular sealing ring 5.

In the embodiment of the present invention, theupper case 22 and thelower case 23 are constructed in a totally enclosed environment and have a snap-fit member that cannot be opened again once being snapped to prevent the contents in thefirst receiving space 223 from being contaminated or leaking.

Needle disk 33 haslancet 331 anddisk 332 perpendicular tolancet 331; the surface of thedisc 332 facing theupper housing 22 has an annular protrusion, and aguide groove 333 engaged with the annular protrusion is fixed inside thesecond receiving space 224 for holding and fixing the needle disc.

As for thetest paper strip 11 related to the invention, it includes having sample pad, colored particle conjugate pad, fibrous membrane and absorbing the filter paper pad according to the order on the liner with non-setting adhesive, the above-mentioned every part overlaps in the part adjacent, there are detection lines and quality control lines on the fibrous membrane, wherein colored particle on the colored particle conjugate pad has anti-A antibody coating, there are anti-B antibody coatings on the detection line, there are anti-A antibodies on the quality control line. The colored particles are colloidal gold particles and the fibrous membrane is typically a nitrocellulose membrane, a nylon membrane, or a polyester membrane.

Example 2

The embodiment of the invention discloses a rapid detection method of an anti-pollution nucleic acid amplification product, which comprises the following steps:

a) taking down the sealing cover;

b) placing the two reaction tubes which are well bathed in the temperature into second containing spaces of the anti-pollution nucleic acid amplification product rapid detection device in the embodiment 1 respectively and fixedly connecting the two reaction tubes on a fixed bracket through a needle head disc; specifically, a PCR reaction tube which is not opened after amplification is placed in a PCR tube hole of a fixed bracket, and the PCR reaction tube comprises a reaction tube and a buffer tube in the embodiment of the invention;

c) pressing the two reaction tubes downwards, and puncturing the bottoms of the reaction tubes by the needle head discs; d) the side wall of the reaction tube is manually extruded, and the reaction liquid passes through the needle disc and is absorbed by the water-guiding fiber membrane to flow into the test strip under the action of gravity and pressure; in other embodiments of the present invention, after the inverted tube body is punctured, the whole sealing device is swung without manually pressing the side wall of the tube body, so that the reaction solution can pass through the needle disc under the action of centrifugal force and be adsorbed and guided by the water-guiding fiber membrane to enter the test strip.

Specifically, by the capillary phenomenon of the water-conducting fiber membrane, the reaction solution moves in the water-conducting fiber membrane and mixes in the sample pad (the extended part of the water-conducting fiber membrane) and slowly moves to the bottom of the nucleic acid detection test strip; covering a sealing cover;

e) detecting the test strip in a visual form through a transparent window and judging and reading the result; the mixed liquid slowly moves from the bottom of the test strip to the top of the nucleic acid detection test strip through capillary phenomenon, and the detection reaction liquid and the detection line on the nucleic acid detection test strip generate antigen-antibody specific reaction to finish the detection process; generally standing for about 10min, and observing at the bottom of the test strip and the part corresponding to the transparent window.

f) After detection, the device is discarded in a safe place as a whole without being opened.

More specifically, the specific detection mechanism of the pollution-prevention fully-closed detection device is as follows: the basic method for converting a target nucleic acid amplification product to be detected into a molecule or molecular complex having immunogenicity is to label a probe with an antigen or hapten (hereinafter, referred to as antigen) and hybridize the labeled specific probe with the target nucleic acid amplification product to be detected, thereby forming a molecular complex having immunogenicity, which can be detected by a method similar to the detection of a protein test strip.

The present invention converts the target nucleic acid amplification product to be detected into molecule or molecular compound with immunogenicity, so that the basic method of protein test paper strip detection technology based on immunoreaction can be applied to nucleic acid test paper strip detection.

Example 3

The invention also discloses the application of the device or the method for quickly detecting the anti-pollution nucleic acid amplification product in the aspects of food industry, agriculture, animal husbandry, customs quarantine inspection, gene mutation detection and DNA single nucleotide polymorphism identification.

It should be noted that, as a general technical platform for closed nucleic acid amplification product detection, the method and the device of the present invention can be widely used in the fields of infectious disease pathogen clinical detection, strategic technical reserve of emergent public health events, agriculture and animal husbandry and food industry, customs inspection and quarantine, environmental detection, biological weapon detection, human genetic disease detection, prenatal and prenatal examination, prenatal and postnatal care, forensic identification, blood margin identification, etc.

Common pathogens include bacteria, fungi, viruses, mycoplasma, chlamydia, parasites, and the like.

The enclosed nucleic acid amplification product detection apparatus can discriminate not only these pathogens but also their subtypes, virulent strains, mutant strains, drug resistance, and the like.

The invention has the following beneficial effects:

1. the anti-pollution nucleic acid amplification product rapid detection device realizes that liquid in the reaction tube flows out to the specified detection card in a completely closed environment, and has the advantages of good closing effect and prevention of primer aerosol pollution;

2. the device and the method for rapidly detecting the anti-pollution nucleic acid amplification product prevent pollution in the detection process due to good sealing performance, can realize rapid real-time detection according to detection and treatment conditions on site or basic level, and have the advantages of convenient operation, simple use and low specialized requirement;

3. the device and the method for quickly detecting the anti-pollution nucleic acid amplification product have the advantages that the device and the method are suitable for quick detection products of new coronary nucleic acid for community and personal field detection, the detection pressure of medical institutions in areas with serious epidemic situations is relieved, and the cross infection risk of examinees is reduced;

4. in the embodiment of the invention, after the inverted tube body is punctured, the whole sealing device is thrown without manually extruding the side wall of the tube body, so that the reaction liquid can also pass through the needle head disc under the action of centrifugal force and be adsorbed and guided by the water guide fiber membrane to enter the test strip.

All the above-mentioned optional technical solutions can be combined arbitrarily to form the optional embodiments of the present invention, and are not described herein again.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. A rapid detection device for anti-pollution nucleic acid amplification products comprises a detection inner core and a sealed outer box, and is characterized in that,

the sealing outer box comprises a sealing cover, an upper shell and a lower shell which are arranged in sequence;

the upper shell comprises an upper end part and a lower end part, the lower end part is buckled with the lower shell to form a first accommodating space, and the detection inner core is arranged in the first accommodating space;

the sealing cover is buckled at the upper end part of the upper shell to form a second accommodating space, an isothermal reaction tube assembly and a needle head disc are arranged in the second accommodating space, and the second accommodating space is used for preventing pollution caused by dropping of the isothermal reaction tube assembly due to device abandonment or mistaken collision of people;

the isothermal reaction tube assembly comprises a tube body and a tube cover which are sequentially connected, and a puncture needle is arranged on the needle head disc; the needle head disc is fixed at a corresponding position below the tube cover, and the puncture needle punctures the tube cover and leads out reaction liquid in the tube body when the isothermal reaction tube assembly moves downwards;

the detection inner core comprises a test strip and a water guide fiber membrane connected with the test strip, and the water guide fiber membrane is arranged at a corresponding position below the needle head disc; when the puncture needle punctures the inverted bottom of the tube body and manually extrudes the side wall of the tube body by downward pressing, the reaction liquid is adsorbed and guided by the water-guiding fiber membrane to enter the test strip through the puncture needle under the action of gravity and pressure, or the reaction liquid is adsorbed and guided by the water-guiding fiber membrane to enter the test strip through the puncture needle under the action of centrifugal force by swinging a sealing device;

and a transparent window is arranged at the upper end part of the upper shell corresponding to the test strip.

2. The device for rapidly detecting the anti-pollution nucleic acid amplification product of claim 1, wherein the test strip is a colloidal gold test strip or a nucleic acid test strip.

3. The device for rapid detection of contamination-resistant nucleic acid amplification product according to claim 1, wherein the tube cover comprises a circular cover body section and a circumferential wall perpendicularly surrounding the circular cover body section; the inner side of the section of the circular cover body is provided with a circular sealing gasket, and when the puncture needle penetrates through the circular sealing gasket, the pipe orifice of the pipe body is abutted against the circular sealing gasket so as to ensure that liquid cannot flow back and cannot flow down by gravity alone.

4. The device for rapid detection of anti-contamination nucleic acid amplification product according to claim 3, further comprising an annular sealing ring disposed between the cap and the tube body, wherein the annular sealing ring is tightly fitted to the edge of the tube opening of the tube body in the radial direction, and a double-layer sealing structure combining a circular sealing gasket and the annular sealing ring is formed between the cap and the tube body, so that the cap does not fall off and liquid flows out due to collision of foreign matter when discarded.

5. The device for rapid detection of anti-contamination nucleic acid amplification product according to claim 4, wherein three layers of closed partitions are alternately arranged on the inner side of the circumferential wall of the tube cover to be tightly pressed with the annular sealing ring.

6. The device for rapid detection of contamination-preventive nucleic acid amplification product according to claim 1, wherein the upper and lower cases are constructed in a totally enclosed environment and have engaging members that cannot be opened once engaged to prevent contamination or leakage of the contents in the first containing space.

7. The rapid antipollution nucleic acid amplification product detection device according to claim 1, wherein the needle disk has a piercing needle and a circular disk perpendicular to the piercing needle; the surface of the disc facing the upper shell is provided with an annular bulge, and a guide groove matched with the annular bulge is fixed in the second accommodating space and used for clamping and fixing the needle head disc.

8. A method for rapidly detecting an anti-contamination nucleic acid amplification product, which is characterized by comprising the following steps:

a) taking down the sealing cover;

b) placing the reaction tube which is well bathed and the hybridization tube which does not need to be bathed into the second containing space of the device for quickly detecting the pollution-proof nucleic acid amplification product of claim 1 respectively and fixedly connecting the reaction tube and the hybridization tube into a guide groove in the upper shell through a needle head disc;

c) pressing the two tube bodies downwards, and puncturing the part with the silica gel cap of the tube cover by the needle head disc;

d) the side wall of the reaction tube and the hybrid liquid tube are manually extruded in sequence, or the rapid detection device is swung, and liquid in the tube drips through a puncture needle of the needle disc head under the action of gravity, pressure or centrifugal force and is adsorbed and guided by a water guide fiber membrane to enter the test strip;

e) the test paper strip is detected through the transparent window in a visual mode, and the result is judged and read.

9. The method for rapid detection of contamination-preventive nucleic acid amplification product according to claim 7, further comprising an operation of covering a sealing cover after the step d);

f) after detection, the device is discarded in a safe place as a whole without being opened.

10. Use of the rapid detection device for contamination-preventive nucleic acid amplification product according to any one of claims 1 to 7 or the rapid detection method for contamination-preventive nucleic acid amplification product according to any one of claims 8 to 9 in the food industry, agriculture, animal husbandry, customs quarantine inspection, gene mutation detection and identification of DNA single nucleotide polymorphism.

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