CN209778863U

CN209778863U - PCR detection kit for detecting heartworm

Info

Publication number: CN209778863U
Application number: CN201920443167.3U
Authority: CN
Inventors: 徐青辰; 罗永村
Original assignee: Bai Sanitary Biotechnology Co Ltd
Current assignee: Bai Sanitary Biotechnology Co Ltd
Priority date: 2019-04-03
Filing date: 2019-04-03
Publication date: 2019-12-13
Anticipated expiration: 2029-04-03

Abstract

the utility model provides a detect heartworm's PCR detect reagent box relates to biological detection technical field, and the main objective is in order to provide a comparatively sensitive heartworm detection mode. The kit comprises a kit body, a lining and a reagent bottle, wherein the kit body is a hexahedral container, the outer surface of the kit body comprises a top side, a bottom side and a peripheral side, and the lining is positioned inside the kit body; the inner liner is provided with a positioning hole, and the reagent bottle is inserted into the positioning hole; the reagent bottle comprises a PCR reaction solution tube, a heat-resistant TaqDNA polymerase solution tube, a forward primer tube, a reverse primer tube, a dNTP tube, a positive control tube and a negative control tube. The method can realize high-sensitivity detection on the heartworm by adopting a PCR detection mode, has extremely high detection speed, and is favorable for early and rapid diagnosis of animals such as dogs infected by the heartworm.

Description

PCR detection kit for detecting heartworm

Technical Field

the utility model belongs to the technical field of the biological detection technique and specifically relates to a PCR detect reagent box for detecting heartworm.

Background

Heartworm (the school name Dirofilaria immitis, english Heartworm) is a linear parasite that spreads with each other via the mosquito bites on pets suffering from Heartworm disease. The larva of heartworm is parasitic in the whole blood, and the adult can grow to 30cm and parasitize in the heart and pulmonary artery. When the heartworms are wound in the heart, the heartworms interfere with blood circulation, so that the heart is hypertrophied and fails, the functions of the lung, the liver and the kidney are further affected, multiple organs fail, and death can be seriously caused. The main host of heartworm is dog, but it also parasitizes in animals such as cat, wolf, suburb, wolf, fox, etc.; in addition, animals such as ferrets and sea lions are also infected with heartworm.

Current diagnostic methods for detecting heartworm include: direct microscopic examination and polypide antigen examination and screening, wherein the microscopic examination is to observe and judge whether the animal is infected with diseases by directly observing the blood of the animal under a microscope, and the polypide antigen screening is to judge whether the animal is infected with heartworm by detecting the antigen in the animal. Both methods require a relatively high or sex-specific population of worms to detect, and are relatively insensitive.

SUMMERY OF THE UTILITY MODEL

An object of the utility model is to provide a PCR detect reagent box that detects heartworm to solve the not sensitive technical problem of heartworm detection technique that exists among the prior art.

In order to achieve the above purpose, the utility model provides a following technical scheme:

The utility model provides a PCR detection kit for detecting heartworm, which comprises a box body, a lining and a reagent bottle, wherein the box body is a hexahedron container, the outer surface of the box body comprises a top side, a bottom side and a peripheral side, and the lining is positioned inside the box body; the inner liner is provided with a positioning hole, and the reagent bottle is inserted into the positioning hole;

The reagent bottle comprises a PCR reaction solution tube, a heat-resistant TaqDNA polymerase solution tube, a forward primer tube, a reverse primer tube, a dNTP tube, a positive control tube and a negative control tube.

The reagent can realize effective amplification of the genetic material of the heartworm by adopting a PCR detection mode, can amplify a detection signal by amplifying limited genetic material, thereby realizing high-sensitivity detection of the heartworm, has extremely high detection speed, and is beneficial to early and fast diagnosis of animals such as dogs infected by the heartworm.

In the above technical solution, preferably, a liquid coolant is disposed between the liner and the box body.

Because the partial reagent that needs when carrying out PCR detection can be better preserve under low temperature environment and not influence its reactivity, and the reagent bottle is inserted and is established on the inside lining, and the inside lining is located the box body, consequently through set up the save reagent that liquid cold-storage agent can be better between inside lining and box body.

In the above technical solution, preferably, the positioning hole is a cylindrical cavity.

In the above technical scheme, preferably, the reagent bottle further comprises a cooling plug-in unit, the cooling plug-in unit is of a hollow structure, a liquid coolant is filled in the cooling plug-in unit, a top surface cavity is arranged at the top of the cooling plug-in unit, and the reagent bottle is connected with the cooling plug-in unit through the top surface cavity and inserted into the positioning hole. Cold-storage agent

the biological activity of the detection reagent can be better kept by adding the cooling insert coolant filled with the liquid coolant.

in the above technical solution, preferably, the cooling insert has a side structure extending downward and forming a bottom surface, and a height of the side surface is smaller than a height of the reagent bottle.

In the above technical solution, preferably, the bottom surface of the cooling insert extends upward to form a bottom surface cavity, and the inner diameter of the bottom surface cavity is the same as that of the cavity located on the top surface of the cooling insert.

In the above technical solution, preferably, the height of the bottom cavity is smaller than the height of the top cavity.

In the above technical solution, preferably, a silica gel ring is disposed on an inner side wall of the cooling insert.

This silica gel circle is convenient for consolidate the reagent bottle to the combination that makes the reagent bottle of placing in the cooling plug-in components and cooling plug-in components is more stable, is difficult for dropping from the cooling plug-in components.

In the above technical solution, preferably, the inner liner is made of a transparent material.

In the above technical solution, preferably, the inner diameters of the positioning holes on different liners are not completely the same.

In the above technical solution, preferably, the coolant is a gel coolant.

In the above technical solution, preferably, the height and the diameter of the reagent bottle are not exactly the same.

Compared with the prior art, the utility model provides a PCR detection kit for detecting heartworm, which comprises a kit body, a lining and a reagent bottle, wherein the kit body is positioned at the outermost side, the lining is positioned inside the kit body, and the reagent bottle is inserted on the lining, thereby realizing the storage of the reagent bottle by the kit body; wherein the inside lining includes dark inside lining and shallow inside lining, and two kinds of inside linings are wrapped up the reagent bottle from the bottom and the top of reagent bottle respectively and are fixed, still have the coolant in inside lining and the box body, and this coolant can make and maintain certain low temperature environment in the box body to guarantee the biological activity of medicine in the reagent bottle.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic diagram of the overall structure of the PCR detection kit for detecting heartworm according to the present invention;

FIG. 2 is a schematic structural view of the liner of FIG. 1;

FIG. 3 is a schematic view of a cooling insert according to the present invention;

FIG. 4 is a schematic view of a first embodiment of the present invention in which the liner and cooling insert are coupled to one another;

FIG. 5 is a schematic cross-sectional view of FIG. 4;

FIG. 6 is a schematic structural view of a second embodiment of the present invention in which the liner and cooling insert are coupled to one another;

FIG. 7 is a cross-sectional schematic view of FIG. 6;

FIG. 8 is a schematic view of a third embodiment of the present invention wherein the liner and cooling insert are coupled to one another;

FIG. 9 is a gel electrophoresis of filaria cordifolia positive and negative controls.

In the figure: 1. a box body; 2. a liner; 21. positioning holes; 3. a reagent bottle; 4. cooling the insert; 41. a top surface cavity; 42. a bottom surface cavity.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. It is to be understood that the embodiments described are only some embodiments of the invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by a person skilled in the art without creative efforts belong to the protection scope of the present invention.

In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in fig. 1, and are only for convenience of description and to simplify the description, but do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore should not be construed as limiting the present invention.

FIG. 1 is a schematic diagram of the overall structure of the PCR detection kit for detecting heartworm according to the present invention; as can be clearly seen from the figures, the device mainly comprises a box body, an inner liner and reagent bottles, wherein the number of the reagent bottles can be increased or decreased according to actual needs.

FIG. 2 is a schematic structural view of the liner of FIG. 1; the upper surface of the lining is provided with a positioning hole, and the section of the positioning hole is circular, so that the positioning hole is convenient to be matched with a cylindrical reagent bottle; when the reagent bottle is designed to be square, the cross section of the positioning hole can be designed to be square correspondingly; in addition, the cross-sectional diameter of the positioning hole is preferably matched with the cross-section of the reagent bottle.

FIG. 3 is a schematic structural view of a cooling insert according to the present invention; the cooling insert may have a variety of configurations, the first being open at the top end only, the cooling insert having a top cavity through which a reagent vial may be inserted into the cooling insert; the other type is that the upper end and the lower end are both provided with openings, the cooling plug-in is provided with a top surface cavity and a bottom surface cavity, and the reagent bottle is inserted into the cooling plug-in through the top end opening.

FIG. 4 is a schematic structural view of a first embodiment of the present invention in which the liner and cooling insert are coupled to one another; as can be seen in this figure, the cooling insert is inserted into the liner through the locating hole.

FIG. 5 is a schematic cross-sectional view of FIG. 4; as can be seen from the figure, the bottom surface and the side surface of the cooling insert are both hollow structures, and the liquid coolant is arranged in the cooling insert.

FIG. 6 is a schematic structural view of a second embodiment of the present invention in which the liner and cooling insert are coupled to one another; as can be seen from the figure, the cooling insert at this point is clearly different from that in fig. 4.

FIG. 7 is a schematic cross-sectional view of FIG. 6; the cooling insert comprises a top surface cavity and a bottom surface cavity, and liquid coolant is also arranged in the surface between the two cavities.

FIG. 8 is a schematic view of a third embodiment of the present invention in which the liner and cooling insert are coupled to one another; when reagent stacks, two inside linings should have at least, and the locating hole that lies in the inside lining of top this moment runs through and connects the upper and lower surface of inside lining, and wherein the inside lining of top lies in below inside lining top to cover the top of the reagent bottle of inserting on the inside lining of below just, thereby realize the low-temperature preservation to the reagent bottle lid, in addition, also can make the reagent bottle that stacks more stable.

FIG. 9 is a gel electrophoresis of filaria cordifolia positive and negative controls; after the corresponding reagent is selected for testing, an accurate detection result can be obtained through an electrophoretogram.

Example 1:

As shown in fig. 1-5, the utility model provides a PCR detection kit for detecting heartworm, comprising a box body 1, a lining 2 and a reagent bottle 3, wherein the box body 1 is a hexahedral container, the outer surface of the box body comprises a top side, a bottom side and a peripheral side, and the lining 2 is positioned inside the box body 1; the inner liner 2 is provided with a positioning hole 21, and the reagent bottle 3 is inserted into the positioning hole 21;

the reagent bottle 3 comprises a PCR reaction solution tube, a heat-resistant TaqDNA polymerase solution tube, a forward primer tube, a reverse primer tube, a dNTP tube, a positive control tube and a negative control tube.

specifically, the negative control tube contains sterile deionized water (ddH)₂O)。

For the convenience of distinction, the positive control tube is set to be red, and the negative control tube is set to be blue. The colors are opposite or other colors can be selected, so long as the function of distinguishing the two control tubes is achieved.

In addition, the concentration of the drug in the forward primer tube and the reverse primer tube used in the reaction is 10 μm, and the corresponding nucleotide sequences are:

the sequence of the positive primer SEQ 01 is 5'-TGATTGGTGGTTTTGGTAA-3'

Reverse primer SEQ 02 sequence 5'-ATAAGTACGAGTATCAATATC-3'

In the positive control tube is the already synthesized heartworm DNA fragment.

Specifically, when the reagent in the present application is used for performing a PCR reaction, the corresponding PCR reaction procedure is as follows: 94 ℃ for 2 min; 94 ℃ 30sec, 53 ℃ 30sec, 72 ℃ 1min, 35 cycles; 10min at 72 ℃; infinity at 4 ℃.

The specific reaction time is about 150 min.

And performing PCR amplification on the sample to be detected according to the mode, wherein if the 689bp fragment is amplified from the sample to be detected, the detection sample contains the heartworm.

When blood of a dog infected with heartworm is taken as a research object, the specific operation process is as follows:

1. nucleic acid extraction

Blood of dogs infected with heartworm was selected as a subject, and DNA was extracted using an automatic Nucleic Acid extractor MagPurix12S and 9.2MagPurix Viral Nucleic Acid Extraction Kit.

The extraction process is as follows: after a reagent pack, disposable plastic products (Reaction chambers, Tip Holder, Piercing Pin, Filtered Tip, Pestle), a Sample tube and an Elute tube are arranged on a laboratory frame, 200 mu L of a Sample is absorbed and injected into the Sample tube, and the Sample tube is placed into an instrument; after pressing the Start key, selecting an extraction program, a sample volume and an extraction volume; after confirming that the correct program is displayed on the LCD screen, pressing an Enter key to start an extraction program; the nucleic acid extracted after the procedure is completed is available for PCR.

2. PCR detection

The total volume of PCR reaction was 25. mu.l, PCR reaction solution was 2.5. mu.l, forward primer was 1. mu.l, reverse primer was 1. mu.l, 10mM dNTP was 2. mu.l, Taq DNA polymerase was 0.5. mu.l, nucleic acid was extracted 5. mu.l, and ddH2O 13. mu.l were extracted.

The PCR reaction program is: 94 ℃ for 2 min; 94 ℃ 30sec, 53 ℃ 30sec, 72 ℃ 1min, 35 cycles; 10min at 72 ℃; infinity at 4 ℃.

3. 2% agar gel electrophoresis of the PCR product

Using a Biomate 100bp DNA Marker, the PCR product was mixed with a DNA fluorescent Dye, subjected to gel electrophoresis, and photographed by a camera system to obtain FIG. 9, in which M represents the Biomate 100bp DNA Marker, (-) represents a negative control, and (+) represents a positive control.

When the test sample detects a band at the same sequence site as the positive control, it means that the dog has been infected with heartworm, otherwise it has no infection.

In addition, in order to ensure the biological activity of the reagents required for the experiment, as an alternative embodiment, a liquid cold-storage agent is arranged between the liner 2 and the box body 1 (the cold-storage agent is provided with an outer package, and the leakage situation can not occur).

Because the partial reagent that needs when carrying out PCR detection can be better preserve under low temperature environment and not influence its reactivity, and reagent bottle 3 is inserted and is established on inside lining 2, and inside lining 2 is located box body 1, consequently through set up the save reagent that liquid cold-storage agent can be better between inside lining 2 and box body 1.

The liquid coolant is a phase-change coolant.

As an alternative embodiment, the positioning hole 21 is a cylindrical cavity.

As an optional embodiment, the reagent bottle further comprises a cooling insert 4, wherein the cooling insert 4 is of a hollow structure, the cooling insert 4 is of a side structure extending downwards and forming a bottom surface, the height of the side surface is smaller than that of the reagent bottle 3, and a liquid coolant is filled in the cooling insert 4; specifically, the top of the cooling insert 4 in this embodiment is provided with a top cavity 41, and the reagent bottle 3 is connected to the cooling insert 4 through the top cavity 41 and inserted into the positioning hole 21.

The biological activity of the detection reagent can be better kept by adding the cooling plug-in 4 filled with the liquid cold-storage agent.

As an alternative embodiment, a silicone ring is provided on the inner side wall of the cooling insert 4.

This silica gel circle is convenient for consolidate reagent bottle 3 to the combination that makes reagent bottle 3 and cooling plug-in components 4 of placing in cooling plug-in components 4 is more stable, is difficult for dropping from cooling plug-in components 4.

As an alternative embodiment, the liner 2 is made of a transparent material.

As an alternative embodiment, the inner diameters of the pilot holes 21 on different liners 2 are not exactly the same.

As an alternative embodiment, the coolant is a gel coolant.

As an alternative embodiment, the height and diameter of the reagent bottle 3 are not exactly the same.

Example 2:

As shown in fig. 6 to 7, the present embodiment 2 is different from embodiment 1 in that: the cooling insert 4 has a top cavity 41, and a bottom cavity 42 is formed by extending the bottom surface of the cooling insert 4 upwards, the bottom cavity 42 has the same inner diameter as the cavity on the top surface of the cooling insert 4 and is disposed corresponding to the cavity up and down, and the specific structure of the cooling insert 4 is shown in fig. 3B.

for more stable fixation of the reagent bottle 3, as an alternative embodiment, the height of the bottom surface cavity 42 is smaller than the height of the top surface cavity 41.

The positioning hole 21 in the liner 2 at this time is arranged on the liner 2 in a penetrating manner and connected with the upper surface and the lower surface of the liner 2, and the liquid coolant is arranged in front of the liner 2 and the box body 1 (the coolant is provided with an outer package, and leakage does not occur), so that the liquid coolant can be placed in the bottom surface cavity 42, and the reagent in the reagent bottle 3 can be stored.

Example 3:

As shown in fig. 8, in the embodiment, the reagent bottles 3 are stacked, and the top surface cavity 41 and the bottom surface cavity 42 of the cooling insert 4 are matched with each other to wrap and cover the reagent bottles 3 from below and above, respectively, so as to further ensure the bioactivity of the medicine in the reagent bottles 3.

Compared with the prior art, the heartworm PCR detection kit has the following advantages:

1. The detection speed is high, the efficiency is high, and pathogens can be detected according to needs.

2. The sensitivity is high, the kit is suitable for early diagnosis, and the detection effect on the canine blood circulation disorder syndrome caused by early heartworm infection is good.

3. the specificity is strong, only specific DNA fragments of heartworm are amplified, and other parasites, bacteria or viruses cannot be amplified; the primer is only specifically combined with the specific DNA fragment of the heartworm, so the specificity is strong and the accuracy is high.

The above description is only for the specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto, and any person skilled in the art can easily think of the changes or substitutions within the technical scope of the present invention, and all should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. the PCR detection kit for detecting the heartworm is characterized by comprising a box body, a lining and a reagent bottle, wherein the box body is a hexahedral container, the outer surface of the box body comprises a top side, a bottom side and a peripheral side, and the lining is positioned inside the box body; the inner liner is provided with a positioning hole, and the reagent bottle is inserted into the positioning hole; the reagent bottle comprises a PCR reaction solution tube, a heat-resistant TaqDNA polymerase solution tube, a forward primer tube, a reverse primer tube, a dNTP tube, a positive control tube and a negative control tube.

2. The PCR detection kit for heartworm according to claim 1, wherein a liquid coolant is provided between the liner and the box body.

3. The PCR detection kit for heartworm according to claim 1, wherein the localization aperture is a cylindrical cavity.

4. the PCR detection kit for heartworm according to claim 1, further comprising a cooling insert, wherein the cooling insert is a hollow structure, the cooling insert is filled with a liquid coolant, and the reagent bottle is connected to the cooling insert and inserted into the positioning hole.

5. The PCR assay kit for heartworm assay according to claim 4, wherein the cooling insert has a side structure extending downward and forming a bottom surface and the height of the side surface is less than the height of the reagent bottle.

6. the PCR detection kit for detecting heartworm according to claim 5, wherein a silica gel ring is arranged on the inner side wall of the cooling insert.

7. The PCR detection kit for detecting heartworm according to claim 1, wherein the inner liner is made of transparent material.

8. The PCR assay kit for heartworm according to claim 1, wherein the inner diameter of the localization hole on different liners is not exactly the same.

9. The PCR detection kit for detecting heartworm according to claim 2, characterized in that the cold-storage agent is a gel cold-storage agent.

10. The PCR detection kit for detecting heartworm according to any one of claims 1 to 9, wherein the height and diameter of the reagent bottle are not exactly the same.