CN111979108A

CN111979108A - Virus conveying and storing tube, manufacturing method and virus conveying and storing method

Info

Publication number: CN111979108A
Application number: CN202010533520.4A
Authority: CN
Inventors: 郭花; 李雷; 曾志奇
Original assignee: ZHIHAI BASO DIAGNOSTICS Inc
Current assignee: GUANGZHOU INSTITUTE OF RESPIRATORY HEALTH; ZHIHAI BASO DIAGNOSTICS Inc.
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2020-11-24

Abstract

The invention discloses a virus conveying and storing tube, a manufacturing method and a virus conveying and storing method, wherein the virus conveying and storing tube comprises a tube body, cell culture solution is stored in the tube body, and a host cell layer consisting of host cells is attached to the inner wall of the tube body; the cell culture solution is used for culturing host cells, and the host cell layer is used for culturing viruses; according to the invention, the virus sample is cultured by the host cell, so that the virus sample is propagated in the conveying process, the probability of missed detection caused by the reduction of the virus titer in the conveying process is reduced, the positive rate of the detected virus is improved, low-temperature storage is not required, and the cost is reduced.

Description

Virus conveying and storing tube, manufacturing method and virus conveying and storing method

Technical Field

The invention relates to the technical field of virus detection, in particular to a virus conveying and storing tube, a manufacturing method and a virus conveying and storing method.

Background

Viruses are an acellular form of nucleic acid molecules (DNA or RNA) and proteins, living by parasitism, an organic species between living and non-living entities. It is composed of a protective shell enclosing a DNA or RNA segment, and is self-replicating by the host cell system via the mechanism of infection, but it cannot grow and replicate independently.

In recent years, more and more new viruses are discovered, such as SARS coronavirus, human highly pathogenic avian influenza virus, new influenza A H1N1 virus, new coronavirus COVID-19, etc., and the emergence of each new virus brings huge harm to human society and also provides a serious challenge for clinical medicine and virology research. And in the face of infectious diseases caused by new viruses, whether accurate laboratory etiology diagnosis can be made in time is the key for effectively controlling epidemic situations. Etiological diagnosis of viruses involves isolation and identification of the virus, determination of infectious units, detection of particles, serological detection, and detection of nucleic acids; the storage and transportation of the virus sample is a very important link in the detection processes, and has a very important influence on the quality of the virus sample.

Currently, the virus detection method is to sample the virus with a sampling swab, and then put the virus into a virus transport preservation solution, such as a virus maintenance solution modified based on a transport medium, and a virus preservation solution modified based on a nucleic acid extraction lysate, for subsequent detection. Although existing virus detection methods use virus-transporting preservative solutions to provide nutrients or reduce factors that degrade viruses and nucleic acids. However, due to the limitations of the fluid components, differences in individual samples, and differences in delivery conditions, viral titers and nucleic acid materials are still susceptible to various reductions during delivery. Meanwhile, the existing virus detection method needs to store the virus at low temperature, and the low-temperature storage and transportation cost is high.

Disclosure of Invention

The first purpose of the invention is to provide a virus transportation and storage tube, which reduces the probability of virus titer and nucleic acid substance reduction during sample transportation.

In order to realize the first purpose of the invention, the invention adopts the following technical scheme:

a virus conveying and preserving tube comprises a tube body, wherein cell culture solution is stored in the tube body, and a host cell layer which consists of host cells and is generated by a monolayer cell culture method is attached to the inner wall of the tube body; the cell culture solution is used for culturing host cells, and the host cell layer is used for culturing viruses.

The virus conveying and storing tube further comprises a screen, wherein the screen is assembled inside the tube body and is arranged at intervals with the inner wall of the tube body; the screen is used to receive a swab stained with a virus sample.

As a specific embodiment, the screen includes a screen mounting portion and a screen body; the outer diameter of the screen mounting part is larger than that of the screen main body, and the screen main body is clamped in the pipe body; a stepped hole for bearing the screen mounting part is formed in the inner wall of the pipe orifice of the pipe body; and sieve pores are formed in the side wall of the sieve mesh main body.

As a specific embodiment, the screen body comprises a first screen section and a second screen section; the first screen part is positioned between the screen mounting part and the second screen part; the screen cloth installation department is the ring-type, first screen cloth portion is cylindricly, second screen cloth portion is big-end-up's coniform.

As a specific embodiment, the stepped bore includes a first cylindrical bore and a second cylindrical bore; the first cylindrical hole is positioned above the second cylindrical hole, the diameter of the first cylindrical hole is larger than or equal to the outer diameter of the screen mounting part, and the diameter of the second cylindrical hole is smaller than the outer diameter of the screen mounting part.

In a specific embodiment, the diameter of the second cylindrical hole is smaller than the inner diameter of the pipe body at the lower side of the pipe orifice.

Further, the virus transportation and preservation tube also comprises a plurality of glass beads or ball bearings which are placed in the screen.

Further, the virus transportation and preservation tube also comprises a tube cover; the pipe cover is sleeved on the pipe orifice of the pipe body and detachably connected with the pipe body.

Further, the virus conveying and preserving tube also comprises a rubber plug; the top end face of the tube cover is provided with a rubber plug mounting hole, and the rubber plug is embedded into the rubber plug mounting hole from the inside of the tube cover.

As a specific implementation mode, the longitudinal section of the rubber plug is in a shape of a Chinese character 'tu', and the rubber plug comprises a first rubber plug part and a second rubber plug part which are arranged from top to bottom; the diameter of the first rubber plug part is equal to the inner diameter of the rubber plug mounting hole; the diameter of the second rubber plug part is equal to the inner diameter of the annular side wall of the pipe cover.

The second purpose of the invention is to provide a method for manufacturing a virus transportation and storage tube, which can reduce the virus titer and the probability of the reduction of nucleic acid substances in the sample transportation process.

In order to achieve the second purpose of the invention, the invention adopts the following technical scheme:

a method for manufacturing a virus transportation and preservation tube is used for manufacturing the virus transportation and preservation tube and comprises the following steps:

unscrewing a tube cover, and adding cell culture solution and host cells into the tube body;

culturing cells by adopting a monolayer cell culture method to ensure that the host cells grow adherently to form compact monolayer host cells;

the screen mesh is arranged in the pipe body, and the pipe cover is screwed tightly.

The third purpose of the invention is to provide a virus transportation and preservation method, which reduces the probability of virus titer and nucleic acid substance reduction during sample transportation.

In order to achieve the third object of the present invention, the present invention adopts the following technical solutions:

the virus conveying and storing method is characterized in that the virus conveying and storing tube is adopted to convey a virus sample, and the method comprises the following steps:

unscrewing a pipe cover, putting the swab into a screen, and screwing the pipe cover;

oscillating the virus conveying and storing tube to elute the virus sample;

and transporting the virus conveying and storing tube to a detection site at normal temperature.

The invention has the beneficial effects that:

the virus conveying and storing tube disclosed by the invention cultures the virus sample through the host cell, realizes the proliferation of the virus sample in the conveying process, reduces the probability of missed detection caused by the reduction of the virus titer in the conveying process, and improves the positive rate of the detected virus. Furthermore, the swab and the inner wall of the tube body are separated by the screen, so that the situation that the swab contacts the inner wall of the tube body and damages a host cell layer attached to the inner wall of the tube body, and viruses cannot invade into the host cell to proliferate, caused by oscillation of the virus conveying and storing tube in the conveying process is avoided. Furthermore, the diameter of the second cylindrical hole is smaller than that of the inner side wall of the pipe body, so that when the screen is inserted into the pipe body, the first screen part and the second screen part of the screen are limited within the range surrounded by the second cylindrical hole and cannot be contacted with the inner side wall of the pipe body, and the situation that the screen is contacted with the inner side wall of the pipe body in the inserting process to damage a host cell layer attached to the inner wall of the pipe body is avoided. Further, the present invention facilitates elution of the virus sample from the swab by multiple frictional contacts of the glass beads with the swab during shaking. Furthermore, the rubber plug capable of being punctured is embedded in the tube cover, so that the material can be injected into the tube body without unscrewing the tube cover. Furthermore, the invention seals through the second rubber plug part, so that the cell culture solution mixed with the virus sample and the host cells in the virus conveying pipe is prevented from leaking in the conveying process, and the aim of safe operation is fulfilled.

Drawings

In order to more clearly illustrate the embodiments of the present invention, the drawings used in the embodiments will be briefly described below. The drawings in the following description are only embodiments of the invention and other drawings can be derived by those skilled in the art without inventive effort.

FIG. 1 is a schematic perspective view of a virus transport and storage tube according to an embodiment of the present invention;

FIG. 2 is a cross-sectional view of a virus transport containment tube according to an embodiment of the present invention;

FIG. 3 is an exploded view of a virus transport containment tube according to an embodiment of the present invention;

FIG. 4 is a cross-sectional view of the tube body of the virus transportation and storage tube according to the embodiment of the present invention;

FIG. 5 is a partial inspection of the host cell layer of a virus delivery storage tube (in a resting state) provided by an embodiment of the invention;

FIG. 6 is a partial view of a layer of host cells in a virus transport storage tube (after shaking) provided by an embodiment of the invention with the screen removed;

FIG. 7 is a partial inspection of the host cell layer of a virus transport storage tube (after shaking) provided by an embodiment of the invention;

FIG. 8 is a flowchart illustrating a method for manufacturing a virus transportation storage controller according to an embodiment of the present invention;

FIG. 9 is a schematic flow chart of a virus transportation and storage method according to an embodiment of the present invention;

description of reference numerals: 100-a pipe body, 110-internal threads, 120-a stepped hole, 121-a first cylindrical hole, 122-a second cylindrical hole, 200-a pipe cover, 210-internal threads, 220-a rubber plug mounting hole, 300-a screen and 310-a screen mounting part; 320-a screen body; 321-a first screen part, 322-a second screen part, 400-a rubber plug, 410-a first rubber plug part, 420-a second rubber plug part.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

As shown in fig. 1 to 3, a virus transportation and preservation tube comprises a tube body 100, a tube cap 200, a screen 300 and a rubber plug 400; the pipe body 100 and the pipe cover 200 are both made of polypropylene materials through surface modification treatment; a host cell layer (not shown) for transporting viruses is attached to the inner wall (including the side inner wall and the bottom inner wall) of the tube 100, a cell culture solution (not shown) for culturing host cells is stored in the tube 100, and the cell culture solution is high enough to soak a swab placed in the tube 100; the host cell layer is a compact monolayer of host cells formed by adherent growth of the host cells under the culture of a monolayer cell culture method; the cell culture solution is used for providing nutrient substances required by the growth of the host cells; the screen 300 is assembled inside the pipe body 100 and spaced apart from the inner wall of the pipe body 100; a receiving space inside the mesh 300 for placing a swab stained with a virus sample; the pipe cover 200 is sleeved on the pipe orifice of the pipe body 100 and detachably connected with the pipe body 100; the top end face of the tube cover 200 is provided with a rubber plug mounting hole 220, and the rubber plug 400 is embedded into the rubber plug mounting hole 220 from the inside of the tube cover 200 and is used for adding lysis solution after being pierced.

In this embodiment, for different virus samples, different types of host cells, such as commercial cells, gene editing cells, or primary cells, can be selected in the tube 100 for culturing, and used for transfection of the virus samples, so that the virus samples can be propagated in the transportation process, the probability of missed detection due to the decrease of virus titer in the transportation process is reduced, and the positive rate of the detected viruses is improved (as shown in tables 1 to 3 below).

In this embodiment, the cell culture solution is prepared from basic powder, HEPES, BSA, phenol red, sucrose, sodium chloride, bacteriostatic agent, etc.

Table 1 shows the results of clinical trials of influenza a specimens transported by the common virus transport storage tube (during the transport of influenza a specimens, no host cells are used to culture influenza a specimens) and the virus transport storage tube provided in this example (during the transport of influenza a specimens, host cells are used to culture influenza a specimens).

As shown in table 1, when the influenza a virus specimen was transported by the ordinary virus transport storage tube, no influenza a positive test could be detected, whereas when the influenza a virus specimen was transported by the virus transport storage tube provided in this example, 4 influenza a positive tests were detected by the colloidal gold test, and the positive test rate was improved by 40%.

Table 2 shows the clinical test results of delivering influenza a virus samples using the common transporting and storing tube for influenza b virus (in the transporting process of influenza b virus samples, host cells are not used to culture influenza b virus samples) and the transporting and storing tube for influenza b virus provided in this example (in the transporting process of influenza b virus samples, host cells are used to culture influenza b virus samples).

As shown in table 2, when the common virus transport storage tube was used to transport the influenza b virus samples, 4 positive cases of influenza b virus were detected by the colloidal gold results, whereas when the virus transport storage tube provided in this example was used to transport the influenza b virus samples, 20 positive cases of influenza b virus were detected by the colloidal gold results, and the detection positive rate was improved by 72%.

Table 3 shows the results of clinical trials of adenovirus samples delivered by the conventional virus delivery storage tube (in the process of delivering adenovirus samples, the host cells are not used for culturing adenovirus samples) and the virus delivery storage tube provided in this example (in the process of delivering adenovirus samples, the host cells are used for culturing adenovirus samples).

As shown in table 3, 48 positive adenovirus samples were detected by colloidal gold results when the adenovirus samples were transported by the ordinary virus transport storage tube, whereas 22 positive adenovirus samples were detected by colloidal gold results when the adenovirus samples were transported by the virus transport storage tube provided in this example, and the detection positivity was improved by 45%.

As shown in tables 1 to 3, the virus transport culture tube provided in this embodiment can perform a sensitization effect on detection results of influenza a, influenza b, and adenovirus, such as qPCR, immunofluorescence, colloidal gold, and the like, thereby increasing the positive rate of detection, and further reducing the rate of missed detection caused by a decrease in virus titer during transport.

In this embodiment, since the virus is propagated during the transportation process, and the normal temperature and the high temperature are more favorable for the virus to infect the host cell, the virus transportation culture tube provided by this embodiment can ensure that the titer of the virus is not reduced without low-temperature cold chain transportation, and the cost is reduced. The cell-containing conveying pipe not only solves the problem that the original virus sample conveying method needs low-temperature cold chain conveying, but also changes the disadvantage of normal temperature or high temperature on sample storage into advantage, and the normal temperature or high temperature conveying is more beneficial to the infection of the virus in the sample on host cells, so that the effect of virus titer proliferation is achieved. Therefore, the titer of the virus sample can not be reduced and can be increased in the transportation process without cold chain conditions, the detection positive rate is finally improved, and the detection omission caused by the reduction of the sample quality is reduced.

In this example, the cell culture medium transport and preservation solution for culturing the host cells was a serum-free medium; the serum-free culture solution does not contain serum and contains various nutrient components (such as growth factors, tissue extracts and the like) which support cell proliferation and biological reaction, so that the virus can be better protected, the influence of the serum on virus infection is reduced, the virus can be favorably infected into host cells, the host cells can be directly inoculated without being washed, and the virus can be conveniently infected into the host cells for proliferation.

As shown in fig. 2 and 4, the outer side wall of the nozzle of the tube body 100 is provided with an external thread 110, the inner side wall of the ring-shaped tube cover 200 is provided with an internal thread 210, and the tube body 100 and the tube cover 200 are detachably connected through a threaded connection, so that the assembly and disassembly are facilitated.

As shown in fig. 2, 3 and 4, the screen 300 includes a screen mounting portion 310 and a screen main body 320 arranged in sequence from top to bottom; the screen body 320 includes a first screen section 321 and a second screen section 322; the mesh mounting part 310 is formed in a ring shape, and is used for clamping the mesh 300 in the pipe body 100; the first screen part 321 is cylindrical, and the second screen part 322 is conical with a large top and a small bottom; the side walls of the first screen part 321 and the second screen part 322 are both provided with screen holes 340 for communicating with the inside of the pipe body 100; the outer diameter of the screen mounting part 310 is larger than that of the first screen part 321, and the outer diameter of the first screen part 321 is equal to that of the top of the second screen part 322; the longitudinal section of the tube body 100 is approximately U-shaped, and the inner wall structure of the tube body 100 is smooth, so that the wall-attached growth of host cells is facilitated; the inner side wall diameter of the tubular body 100 is greater than the outer diameter of the first screen section 321; a stepped hole 120 for receiving the screen mounting part 310 is formed in the inner side wall of the nozzle of the pipe body 100; the stepped hole 120 comprises a first cylindrical hole 121 and a second cylindrical hole 122, the first cylindrical hole 121 is positioned above the second cylindrical hole 122, the diameter of the first cylindrical hole 121 is larger than the outer diameter of the screen mounting part 310, and the outer diameter of the screen mounting part 310 is larger than the diameter of the second cylindrical hole 122; the stepped surface formed between the first cylindrical bore 121 and the second cylindrical bore 122 is adapted to receive a screen mounting portion 310.

In this embodiment, the virus transport and storage tube further comprises glass beads (not shown) for eluting the virus sample, the glass beads being disposed in the screen 300; after the swab is placed in the screen 300, the tube cap 200 is first screwed down, and then the virus carrying tube is shaken, and the glass beads are in multiple frictional contact with the swab in the shaking process, so that the virus sample on the swab is conveniently eluted.

As shown in fig. 2 and 4, in the present embodiment, the diameter of the inner sidewall of the tube 100 is larger than the diameter of the second cylindrical hole 122, so that when the screen 300 is inserted into the tube 100, the first screen portion 321 and the second screen portion 322 of the screen 300 are limited within the range surrounded by the second cylindrical hole 122, and will not contact with the inner sidewall of the tube 100, thereby avoiding the damage to the host cell layer attached to the inner wall of the tube 100 due to the contact with the inner sidewall of the tube 100 during the insertion of the screen 300.

In this embodiment, after the swab stained with the virus sample is placed in the accommodating space inside the screen 300, the isolation effect of the screen 300 does not contact the inner wall of the tube 100, so as to prevent the virus from shaking during transportation and storage, and the swab shakes and contacts the inner wall of the tube 100, thereby damaging the host cell layer adhered to the inner wall of the tube 100, and causing the virus to be unable to invade the host cell for proliferation.

As shown in fig. 5, the swab is placed in a virus conveying and storing tube, and after standing for a period of time at the temperature of 2-8 ℃, the cells are seen to be dense and full in the visual field, the boundary is clear, and the adherence is good; as shown in fig. 6, the virus transporting and preserving tube without the screen and with the swab placed therein was subjected to low-speed shaking by using a shaker, and after 4 hours, it was seen that the cells were detached and suspended in the visual field, and partially broken, and a large amount of cells were fragmented; as shown in FIG. 7, the virus-transporting and storing tube with the mesh and the swab placed therein was shaken at a low speed by a shaker, and after 4 hours, it was observed that the cells remained almost in the same state as in the standing state in the visual field, and a very small amount of the cells fell off.

As shown in fig. 3, the longitudinal section of the rubber plug 400 is in a shape of a Chinese character 'tu', and comprises a first rubber plug part 410 and a second rubber plug part 420 which are arranged from top to bottom; the diameter of the first rubber plug part 410 is equal to the inner diameter of the rubber plug mounting hole 220, and is used for being embedded into the rubber plug mounting hole 220, so that the rubber plug 400 is fixed on the tube cover 200, and the lysate can be conveniently added after being pierced; the diameter of the second packing part 420 is equal to the inner diameter of the annular sidewall of the cap 200 for sealing.

In this embodiment, due to the sealing effect of the second rubber plug part 420, the virus transporting and storing tube has good sealing performance, so that the cell culture fluid mixed with the virus sample and the host cells in the virus transporting tube is prevented from leaking during the transporting process, and the purpose of safe operation is achieved; when the detector needs to inject other substances, such as lysate, into the virus conveying and storing tube, the injection can be completed only by puncturing the rubber plug 400 with the syringe without opening the tube cap 200.

In this example, the lysate is prepared from a denaturant, a surfactant, a protease, a chelating agent, and the like.

As shown in fig. 8, a method for manufacturing a virus transportation storage tube includes the following steps:

culturing cells by adopting a monolayer cell culture method to ensure that the host cells grow adherently to form a compact host cell layer;

As shown in fig. 9, a virus transportation and preservation method includes the following steps:

oscillating the virus conveying and storing tube to elute the virus sample;

In this embodiment, the virus transportation and storage method further comprises attaching a label to the virus transportation and storage tube, and labeling specimen information on the label for management; the virus conveying and storing tube is transported to a detection place at normal temperature, such as a laboratory, and then can be directly detected or heated to 37 ℃ for 12-48 hours; if a lysing treatment is desired, the tube can be filled with the lysate by puncturing the plug.

The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the present invention may occur to those skilled in the art without departing from the principle of the present invention, and such modifications and embellishments should also be considered as within the scope of the present invention.

Claims

1. The utility model provides a virus transport preservation pipe, includes the body, its characterized in that: the cell culture solution is stored in the tube body, and a host cell layer consisting of host cells is attached to the inner wall of the tube body; the cell culture fluid is used for culturing host cells, and the host cell layer is used for culturing viruses.

2. The virus transport and storage tube of claim 1, wherein: the screen is assembled inside the pipe body and is arranged at intervals with the inner wall of the pipe body; the screen is used to contain a swab stained with a viral sample.

3. The virus transport and storage tube of claim 2, wherein: the screen comprises a screen mounting part and a screen main body; the outer diameter of the screen mounting part is larger than that of the screen main body, and the screen main body is clamped in the pipe body; a stepped hole for bearing the screen mounting part is formed in the inner wall of the pipe orifice of the pipe body; and sieve pores are formed in the side wall of the sieve mesh main body.

4. The virus transport and storage tube of claim 3, wherein: the screen body comprises a first screen section and a second screen section; the first screen part is positioned between the screen mounting part and the second screen part; the screen cloth installation department is the ring-type, first screen cloth portion is cylindricly, second screen cloth portion is big-end-up's coniform.

5. The virus transport and storage tube of claim 3, wherein: the stepped hole comprises a first cylindrical hole and a second cylindrical hole; the first cylindrical hole is positioned above the second cylindrical hole, the diameter of the first cylindrical hole is larger than or equal to the outer diameter of the screen mounting part, and the diameter of the second cylindrical hole is smaller than the outer diameter of the screen mounting part.

6. The virus transport and storage tube of claim 5, wherein: the diameter of the second cylindrical hole is smaller than the inner diameter of the pipe body at the lower side of the pipe orifice adjacent to the pipe orifice.

7. The virus transport and storage tube according to any one of claims 2 to 6, wherein: also comprises a plurality of glass beads or rolling balls; the glass beads or balls are placed on the screen.

8. The virus transport and storage tube according to any one of claims 1 to 6, wherein: the device also comprises a tube cover; the pipe cover is sleeved on the pipe orifice of the pipe body and detachably connected with the pipe body.

9. The virus transport and storage tube of claim 8, wherein: also comprises a rubber plug; the top end face of the tube cover is provided with a rubber plug mounting hole, and the rubber plug is embedded into the rubber plug mounting hole from the inside of the tube cover.

10. The virus transport and storage tube of claim 9, wherein: the longitudinal section of the rubber plug is in a convex shape and comprises a first rubber plug part and a second rubber plug part which are arranged from top to bottom; the diameter of the first rubber plug part is equal to the inner diameter of the rubber plug mounting hole; the diameter of the second rubber plug part is equal to the inner diameter of the annular side wall of the pipe cover.

11. A method for manufacturing a virus transportation/storage tube according to any one of claims 8 to 10, comprising the steps of:

12. A method for transporting and preserving viruses, which is characterized in that the virus transporting and preserving tube of any one of claims 8-10 is used for transporting virus samples, and comprises the following steps:

oscillating the virus conveying and storing tube to elute the virus sample;