CN113736868A

CN113736868A - RNA nucleic acid detection reagent storage method, kit and use method

Info

Publication number: CN113736868A
Application number: CN202111084753.1A
Authority: CN
Inventors: 陈伟珊; 孙江山; 吴纯阳
Original assignee: Guangzhou Shengling Medical Technology Co ltd
Current assignee: Guangzhou Shengling Medical Technology Co ltd
Priority date: 2021-09-16
Filing date: 2021-09-16
Publication date: 2021-12-03
Anticipated expiration: 2041-09-16
Also published as: CN113736868B

Abstract

The invention discloses a preservation method of a RNA nucleic acid detection reagent, a kit and a use method, wherein the preservation method comprises the steps of packaging a first mixture, a second mixture and a third mixture in a reagent container, and separating the first mixture from the second mixture through a paraffin wax sheet; the other side of the second mixture is separated from the third mixture by a paraffin plate. The kit comprises a tube body, a first paraffin sheet and a second paraffin sheet; a first mixture is packaged between the first paraffin sheet and the opening of the pipe body; a second mixture is packaged between the first paraffin sheet and the second paraffin sheet; and a third mixture is encapsulated between the second paraffin sheet and the closed end of the tube body. Different components of the reagent are separated by paraffin, so that the components of the reagent can be stably stored for a long time at normal temperature, and the detection effect is ensured not to be reduced; through the dilatation to the reagent pipe, conveniently mix the reagent and evenly.

Description

RNA nucleic acid detection reagent storage method, kit and use method

Technical Field

The invention relates to a preservation method of an RNA nucleic acid detection reagent, a kit formed according to the method and a practical method of the kit.

Background

The RNA nucleic acid detecting reagent is a technology of reverse transcribing RNA into cDNA and detecting specific gene.

The RNA nucleic acid detection reagent has the advantages that a plurality of enzymes and a plurality of substrates are combined for use, the preservation period of the enzyme and the substrate after mixing is usually short, and the enzymes can easily inhibit each other, for example, taq enzyme can inhibit the activity of reverse transcriptase. In order to avoid the inhibition of DNA polymerase activity by reverse transcriptase, the skilled worker generally has the following measures: (1) reverse transcription and PCR amplification are carried out in two steps, usually, reverse transcription is firstly carried out to directly inactivate reverse transcriptase so as to avoid inhibiting the activity of DNA polymerase; (2) after reverse transcription is finished, the inhibition level of reverse transcriptase to DNA polymerase is reduced by phenol extraction and ethanol precipitation of reverse transcription products; (3) adding non-homologous RNA as a vector to reduce the level of inhibition of DNA polymerase by reverse transcriptase; (4) adding exogenous DNA; (5) the T4 gene 32 protein was added to reduce the level of DNA polymerase inhibition by reverse transcriptase. In the method, the steps (1) and (2) are completed through multiple steps, the process is relatively complicated, and new unpredictable problems are easily introduced; (3) the method (5) is to add some proteins or nucleic acids which are not necessary for the reaction, the added proteins and nucleic acids are expensive, and the chemical activity of the added proteins and nucleic acids is not particularly stable, and thus the desired purpose cannot be achieved easily due to the problem of the addition.

How to use fewer steps and lower the cost and report that all components of the detection reagent are stable and effective for a long time is a problem to be solved urgently.

Disclosure of Invention

The embodiment of the application provides a preservation method, a kit and a use method of an RNA nucleic acid detection reagent, solves the problems that in the prior art, enzymes are easy to inhibit each other when multiple enzymes and substrates are combined for use, the steps of the prior art are complex, the cost is high, and the components of the reagent are unstable, and realizes the effects of long preservation time, stability and concise use steps of the components of the reagent.

The embodiment of the application provides a preservation method of an RNA nucleic acid detection reagent, which comprises the steps of mixing reverse transcriptase, 50% of glycerol and an RNA inhibitor to prepare a first mixture;

mixing the downstream primer, the detection probe and the amplification reaction solution to prepare a second mixture;

mixing Taq DNA polymerase, an upstream primer and a reaction solution to prepare a third mixture,

the first mixture, the second mixture and the third mixture are packaged in a reagent container, and the first mixture and the second mixture are separated by a paraffin slice;

the other side of the second mixture is separated from the third mixture by a paraffin plate;

the melting point temperature of the paraffin sheet between the first mixture and the second mixture is lower than the melting point temperature of the paraffin sheet between the second mixture and the third mixture.

Further, the melting point temperature of the paraffin wax sheet between the first mixture and the second mixture is 36-38 ℃;

the melting point temperature of the paraffin wax sheet between the second mixture and the third mixture is 48-52 ℃.

A kit comprises a tube body, a first paraffin sheet and a second paraffin sheet;

the first paraffin sheet is fixed in the tube body, and the edge of the first paraffin sheet is in liquid seal with the inner wall of the tube body;

the second paraffin sheet is fixed in the tube body, and the edge of the second paraffin sheet is in liquid seal with the inner wall of the tube body;

the first paraffin wax sheet and the second paraffin wax sheet are arranged at intervals;

a first mixture prepared by mixing reverse transcriptase, glycerol and an RNA inhibitor is packaged between the first paraffin sheet and the opening of the tube body;

a second mixture prepared by mixing a downstream primer, a detection probe and an amplification reaction solution is encapsulated between the first paraffin sheet and the second paraffin sheet;

and a third mixture prepared by mixing Taq DNA polymerase, an upstream primer and reaction liquid is packaged between the second paraffin sheet and the closed end of the tube body.

Further, the sealing device also comprises a sealing component, wherein the sealing component comprises a fixing ring, a rubber sleeve and an end cover;

the fixing ring is used for being detachably, hermetically and fixedly connected with the opening end of the pipe body;

the two ends of the rubber sleeve are provided with openings and are used for forming an elastic pipe body expansion space, so that different mixtures can be fully mixed;

an opening at one end of the rubber sleeve is fixedly communicated with the top end of the fixing ring in a sealing manner;

the end cover is used for drawing the rubber sleeve and changing the shape of the rubber sleeve, the other end of the rubber sleeve is fixed with the bottom surface of the end cover in a sealing mode, the bottom surface of the end cover is connected with the fixing ring through a buckle, and the rubber sleeve extends into the pipe body when the end cover is fixed with the fixing ring through the buckle.

Further, the closure assembly further comprises an adjustment assembly;

the adjusting assembly is used for forming holes on the paraffin sheet or breaking the paraffin sheet;

the adjusting assembly comprises a rotating part, a connecting shaft, a sleeve, a telescopic rod and a fixing rod;

the rotating part is positioned on the outer side of the end cover and fixedly connected with the connecting shaft, and the connecting shaft penetrates through the end cover and is fixedly connected with the sleeve; the sleeve is a cylindrical barrel, and one end of the cylindrical barrel, which is far away from the connecting shaft, is opened; the telescopic rod extends into the sleeve, and the telescopic rod is in threaded fit with the inner wall of the sleeve;

the telescopic rod is provided with an axial elongated slot, one end of the fixed rod is fixed at the edge of the opening of the pipe body, and the other end of the fixed rod extends into the elongated slot, so that the telescopic rod can move along the axial direction when the sleeve rotates.

Further, rotating part center department, connecting axle, sleeve inner wall, telescopic link are located the end and the telescopic link inner core and the end of sleeve and are made by metal, and the end of flexible pipe can rapid heating up when heating rotating part center department.

Further, the paraffin piece is funnel-shaped or toper, and its tip department is closed, and the tip is towards the body open end.

Furthermore, the edge of the opening end of the paraffin plate is provided with a circular paraffin ring with a symmetrical longitudinal section;

a baffle ring is fixed at the positioning positions of the first paraffin sheet and the second paraffin sheet in the pipe body;

the paraffin ring is characterized in that the retaining ring is frustum-shaped, one end with the large diameter is fixedly connected with the inner wall of the pipe body in a sealing mode, and one end with the small diameter of the retaining ring faces the opening side of the pipe body, so that molten paraffin liquid can enter a gap between the retaining ring and the pipe body when the paraffin ring is molten.

A kit using method, comprising the kit of claim 4, and comprising the following specific steps:

1) separating the end cover from the fixing ring;

2) heating to melt the first paraffin wax sheet, enabling the opening end of the pipe body to face downwards, and repeatedly pulling the rubber sleeve to quickly and uniformly mix the first mixture and the second mixture;

3) and heating to melt the second paraffin wax sheet, and repeatedly pulling the rubber sleeve to uniformly mix the first mixture, the second mixture and the third mixture.

A method for using the kit, comprising the kit according to claim 8, comprising the following steps:

1) separating the end cover from the fixing ring;

2) rotating the rotating part to enable the end head of the telescopic rod to abut against the tip end of the paraffin sheet, directly puncturing the tip end of the first paraffin sheet or heating the end head of the telescopic rod through a constant-temperature heating device, and melting paraffin to form a hole when the temperature of the end head reaches the melting temperature of the paraffin sheet;

the first mixture and the second mixture are quickly and uniformly mixed by repeatedly pulling the rubber sleeve;

3) repeating the step 2) to form a hole at the tip of the second paraffin plate; and repeatedly pulling the rubber sleeve to uniformly mix the first mixture, the second mixture and the third mixture. .

One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages: 1) different components of the reagent are separated by paraffin, so that the components of the reagent can be stably stored for a long time at normal temperature, and the detection effect is ensured not to be reduced;

2) through the dilatation to the reagent pipe, conveniently mix the reagent and evenly.

Drawings

FIG. 1 is a schematic structural view of the present invention;

FIG. 2 is a schematic diagram of the construction of the kit;

FIG. 3 is a schematic view of the open end cap of the cartridge;

FIG. 4 is a schematic diagram of a cartridge with an adjustment assembly;

FIG. 5 is a schematic view of the adjustment assembly;

FIG. 6 is a schematic view of a paraffin plate structure;

FIG. 7 is a schematic view of a retainer ring structure;

FIG. 8 is a schematic view showing a state of use of the kit;

FIG. 9 is a schematic view showing a state where a mixture in the reagent cartridge is mixed;

FIG. 10 is an amplification map;

FIG. 11 is a graph of amplification reaction profiles for different pre-denaturation times;

FIG. 12 double-layer sub-paraffin amplification profile;

FIG. 13 amplification profile without paraffin addition.

In the figure, a pipe body 10, a baffle ring 11, a first paraffin sheet 20, a second paraffin sheet 30, a closing component 40, a fixed ring 41, a rubber sleeve 42, an end cover 44, an adjusting component 43, a rotating part 431, a connecting shaft 432, a sleeve 433 and a telescopic rod 434; a paraffin ring 50.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like as used herein are for illustrative purposes only and do not represent the only embodiments.

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 invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The RNA nucleic acid detection reagent contains reverse transcriptase, DNA polymerase, reverse transcription primers, nucleic acid amplification substrates dntps (or dutps) and reaction buffer solution, and the like, and before nucleic acid amplification, inert chemical components are used for physically separating multiple components necessary for nucleic acid amplification, so that performance reduction caused by mixing and storing the components is avoided. The components which are easy to react are combined through the paraffin flakes, so that the long-time stability of the components stored at normal temperature is realized, and the performance of the reagent can not be reduced when the reagent is stored at normal temperature for a long time.

Example one

As shown in FIG. 1, a preservation method of an RNA nucleic acid detection reagent comprises mixing reverse transcriptase with 50% glycerol and an RNA inhibitor to prepare a first mixture;

In actual production, the melting point temperature of the paraffin wax sheet between the first mixture and the second mixture is 36-38 ℃;

The actual test was performed by amplification of the detection reagents by taq-man probe method of sindbis virus: the detection reagent preservation method aims at the target of the conservation area of the Sindbis virus and comprises the following steps:

an upstream primer: CGACAGTGAGAACAGCCAGA

A downstream primer: CCTACTTTCATCGCGGCAGT

And (3) probe: AGGCGTACGTCGAATTGTCAGCA

The Sindbis virus amplification target nucleotide sequence is as follows:

TTCATGTGGGGAGGAGCACAATGTTTTTGCGACAGTGAGAACAGCCAGATGAGTGAGGCGTACGTCGAATTGTCAGCAGATTGCGCGACTGACCACGCGCAGGCGATTAAGGTACATACTGCCGCGATGAAAGTAGGACTGCGTATAGTG

experiment one, the Sindbis virus Taq-man probe method amplification detection reagent comprises an amplification reaction solution, Taq DNA polymerase, reverse transcriptase, an upstream primer, a downstream primer, a probe, DEPC-H2O and a small chamber prepared from n-hexacosane and n-eicosane through a specific process.

The small chamber formed by the n-hexacosane and the n-eicosane divides the whole reaction system into an upper, a middle and a lower independent small chambers, and the lower small chamber contains Taq DNA polymerase and an upstream primer; the middle chamber contains a reverse transcription downstream primer, a detection probe and an amplification reaction solution; the upper chamber contains reverse transcriptase, rnase inhibitor. The reaction components and paraffin profile are shown in FIG. 1.

Effect of different volumes of bottom Paraffin on amplification reactions

1. Under the condition of keeping the volumes of the reaction system and the paraffin wax at the upper layer unchanged, the volume of the paraffin wax at the lower layer is changed, and the adding amounts of the paraffin wax are respectively 0ul, 10ul, 15ul, 20ul and 25 ul. The same PCR template was used for amplification and the amount of paraffin was determined by a plot of the fluorescent quantitative PCR instrument ABI7500 amplification. The configuration is as in table one:

	1	2	3	4	5
						PCR reaction system	20ul	20ul	20ul	20ul	20ul
Top layer paraffin	15ul	15ul	15ul	15ul	15ul
						Lower layer paraffin wax	0ul	10ul	15ul	20ul	25ul

The volume of each component in the PCR reaction system is shown in Table II.

Watch two

Reagent	Volume (ul)
		10X Buffer	2.5
dNTP	0.1
		Primer F	0.1
Primer R	0.1
		Probe needle	0.05
Reverse transcriptase	0.05
		RNase inhibitors	0.2
taq enzyme	0.5
		50 glycerin	0.25
Sterilized DEPC-water	16.15
		Total up to	20

2. PCR amplification conditions (see Table III)

Watch III

3. Conclusion

The effect of paraffin wax addition on the CT value of the PCR reaction is shown in Table four, and the amplification map is shown in FIG. 10.

Watch four

Lower paraffin volume (ul)	CT1	CT2	CT3	Mean value of
					0	23.13	23.17	23.20	23.17
10	23.32	23.50	23.68	23.50
					15	23.61	23.77	23.28	23.55
20	23.15	23.15	23.39	23.23
					25	23.24	23.44	23.34	23.34

As can be seen from the first and fourth tables and FIG. 10, the amplification curve with paraffin was not significantly different from that without paraffin. Paraffin less than 10ul cannot completely cover the lower chamber, so that the isolation effect cannot be achieved, and the paraffin is not included in the test range.

Experiment two, the effect of different pre-denaturation times on the amplification reaction was tested, and the results are as follows

1. Under the condition of keeping the volume of each component and paraffin unchanged, performing gradient dilution on the male die, diluting by the power of N of 10 to 5 continuous gradients, and changing the pre-denaturation time, wherein the pre-denaturation time is 3min, 10min and 15min respectively. The effect of different pre-denaturation times on the amplification curve was observed using fluorescent quantitative PCR instrument ABI7500 amplification. The amplification procedure is shown in Table five.

Watch five

Procedure	Reverse transcription	Pre-denaturation	PCR
				1	50℃：30min	95℃：3min	(95 ℃ C.: 5s, 55 ℃ C.: 60 s) X45 cycle
2	50℃：30min	95℃：10min	(95 ℃ C.: 5s, 55 ℃ C.: 60 s) X45 cycle
				3	50℃：30min	95℃：15min	(95 ℃ C.: 5s, 55 ℃ C.: 60 s) X45 cycle

2. After amplification by a PCR instrument, an amplification curve is observed, and paraffin is found to cause instability in the early stage of a baseline, so that the curve graph is inclined to different degrees. The baseline range is reset, and after the baseline is corrected, the difference of the graphs of the three programs is not large. The curve of procedure 3 had the smallest difference before and after baseline adjustment, procedure 3 was selected as the amplification procedure, and the amplification curve is shown in FIG. 11.

Experiment three, through experiment one and experiment two, select 10ul bottom paraffin, 15min pre-denaturation time, make the double-deck paraffin reaction of Sindbis virus and not add paraffin reaction and compare, the result is shown in figure four, figure five.

Experiment three

Through the first experiment and the second experiment, 10ul of bottom paraffin is selected, the pre-denaturation time is 15min, the double-layer paraffin reaction of the Sindbis virus is compared with the reaction without paraffin, and the result is shown in figure 12 and figure 13.

As shown in the fourth and fifth graphs, the CT of each gradient of the amplification curve of the double-layer paraffin and the amplification curve without paraffin is not obviously different, and the paraffin has no influence on PCR amplification.

The Sindbis virus Taq-man probe method amplification detection reagent prepared by the method detects artificially synthesized RNA target fragments, and directly mixes an amplification reaction solution, Taq DNA polymerase, reverse transcriptase, an upstream primer, a downstream primer, a probe and DEPC-H2O by a common method for detection, the detection result prepared by the method is compared with the common method, the CT value of the patent method is not obviously different from the CT value of the common method when the fluorescence PCR amplification detection result is immediately prepared, the detection result of a low-concentration sample (500 copies/ml) patent method is 100 percent (20/20), the detection result of the common method is 50 percent (10/20), and the detection result is stored at-20 ℃ for one month after the preparation.

The comparison shows that the method improves the storage stability of the reagent and improves the detection sensitivity of the reagent

Example two

As shown in fig. 2-3, a kit comprises a tube 10, and further comprises a first paraffin sheet 20 and a second paraffin sheet 30;

the first paraffin plate 20 is fixed in the pipe body 10, and the edge of the first paraffin plate 20 is in liquid seal with the inner wall of the pipe body 10;

the second paraffin plate 30 is fixed in the pipe body 10, and the edge of the second paraffin plate 30 is in liquid seal with the inner wall of the pipe body 10;

the first paraffin wax sheet 20 and the second paraffin wax sheet 30 are arranged at intervals;

a first mixture prepared by mixing reverse transcriptase, 50% glycerol and an RNA inhibitor is packaged between the first paraffin plate 20 and the opening of the tube body 10;

a second mixture prepared by mixing a downstream primer, a detection probe and an amplification reaction solution is encapsulated between the first paraffin sheet 20 and the second paraffin sheet 30;

a third mixture prepared by mixing Taq DNA polymerase, an upstream primer and a reaction solution is encapsulated between the second paraffin plate 30 and the closed end of the tube body 10.

Therefore, different components of the reagent are isolated, and the stability of each component and the stability of the final detection effect can be ensured when the reagent is stored at normal temperature.

EXAMPLE III

In practice, the reagent is usually packaged in a relatively small tube 10, the mixing space of the components is very small, and before mixing, the paraffin is melted or punctured, so that the operation space is limited. And the fragments can float on the liquid surface after the paraffin is melted or punctured, so that stirring and mixing are inconvenient.

Thus, a closure assembly 40 is added, which comprises a securing ring 41, a gum cover 42 and an end cap 44;

the fixing ring 41 is used for being detachably, hermetically and fixedly connected with the opening end of the pipe body 10;

the two ends of the rubber sleeve 42 are open and used for forming an elastic expansion space of the pipe body 10, so that different mixtures can be fully mixed;

an opening at one end of the rubber sleeve 42 is fixedly communicated with the top end of the fixing ring 42 in a sealing manner;

the end cover 44 is used for pulling the rubber sleeve 42 and changing the shape of the rubber sleeve 42, the other end of the rubber sleeve 42 is fixed with the bottom surface of the end cover 44 in a sealing mode, the bottom surface of the end cover 44 is connected with the fixing ring 41 through a buckle, and the rubber sleeve 42 extends into the pipe body 10 when the end cover 44 is fixed with the fixing ring 41 through the buckle.

An expanded space is formed by the closure assembly 40 to allow for rapid mixing of the reagents even if the paraffin is not completely melted. And the deformation of the rubber sleeve 42 can facilitate the impact on the reagent liquid, and the rapid and uniform mixing is facilitated.

The sealing component 40 can be made of disposable materials such as medical plastics, is simple and convenient, expands the volume of the tube body 10 and facilitates the mixing operation of reagents.

Example four

As shown in fig. 3 to 9, when the paraffin wax is actually melted at different temperatures, it is found that waiting time is relatively long by sequentially melting the first paraffin wax sheet 20 and the second paraffin wax sheet 30 at different temperatures. And the paraffin wax is heated, the outer edge of the paraffin wax is melted first, and the rest part of the paraffin wax integrally floats to the liquid surface. Under the condition that can carry out the dilatation, if in the space after the dilatation can be flowed to liquid, mix, paraffin does not influence the mixing process, and the operation is received the influence and is littleer.

The closure assembly 40 further includes an adjustment assembly 43;

the adjusting assembly 43 is used for forming holes on the paraffin sheet or breaking the paraffin sheet;

the adjusting assembly 43 comprises a rotating part 431, a connecting shaft 432, a sleeve 433, a telescopic rod 434 and a fixed rod 435;

the rotating part 431 is positioned outside the end cover 44, the rotating part 431 is fixedly connected with a connecting shaft 432, and the connecting shaft 432 passes through the end cover 44 and is fixedly connected with a sleeve 433; the sleeve 433 is a cylindrical tube and is open at one end far away from the connecting shaft 432; the telescopic rod 434 extends into the sleeve 433, and the telescopic rod 434 is in threaded fit with the inner wall of the sleeve 433;

an axial elongated slot is formed in the telescopic rod 434, one end of the fixed rod 435 is fixed to the edge of the opening of the pipe body 10, and the other end of the fixed rod extends into the elongated slot, so that the telescopic rod 434 can move axially when the sleeve 433 rotates.

The telescopic rod 434 is used for perforating the paraffin piece, so that the reagent liquid is mixed and flows into the expanded space when the pipe body 10 is inverted, and the swinging rubber sleeve 42 is repeatedly pulled and pulled to realize the uniform mixing of the reagent liquid. The position of the paraffin plate is also positioned on the tube body 10, and the problem of melting the paraffin integrally does not need to be considered.

When the telescopic rod 434 is used for melting paraffin sheets, the center of the rotating part 431, the connecting shaft 432, the inner wall of the sleeve 433, the end of the telescopic rod 434 located in the sleeve 433, and the inner core and the end of the telescopic rod 434 are made of metal, so that the end of the telescopic rod 434 can be rapidly heated when the center of the rotating part 431 is heated. The utility model has the advantages of can be used homothermal electric rod to heat rotary part 431 center metal position, and then make the end heat up to paraffin melting's temperature.

EXAMPLE five

The flat paraffin plate is easy to crack integrally when being punctured, and can be mixed into reagent liquid to interfere subsequent mixing and detection operation. Thus, the paraffin wax chip is arranged in a funnel or cone shape with its tip closed and the tip facing the open end of the tube 10.

Thus, after the tip is punctured, the reagent liquid in the chamber partitioned by the inverted tube 10 can smoothly flow into the expanded space. There is no need to completely melt or break the paraffin flakes. Furthermore, the melting points of the paraffin pieces can be the same, and the paraffin pieces with different melting points do not need to be selected. Nor is it necessary to completely melt the paraffin flakes prior to the mixing operation.

The installation process of the paraffin plate is troublesome, and the edge of the opening end of the paraffin plate is arranged to be provided with a circular paraffin ring 50 with a symmetrical longitudinal section; as shown in fig. 6-7.

A baffle ring 11 is fixed at the positioning positions of the first paraffin plate 20 and the second paraffin plate 30 in the pipe body 10;

the blocking ring 11 is frustum-shaped, one end with the large diameter is fixedly connected with the inner wall of the pipe body 10 in a sealing mode, one end with the small diameter of the blocking ring 11 faces the opening side of the pipe body 10, and molten paraffin liquid can enter a gap between the blocking ring 11 and the pipe body 10 when the paraffin ring 50 is molten.

During actual assembly, the baffle ring 11 is fixed at the reagent liquid separation area, then the paraffin plate slides to the baffle ring 11, the pipe body 10 at the position of the outer circumference of the baffle ring 11 is heated, paraffin is melted and flows into a gap between the baffle ring 11 and the pipe body 10, and the fixation of the paraffin plate and the separation of the reagent liquid are realized.

EXAMPLE six

The use method of the kit comprises the three kits of the embodiments, and the specific steps are as follows:

1) separating the end cap 44 from the fixing ring 41;

2) heating to melt the first paraffin wax sheet 20, placing the open end of the pipe 10 downwards, and repeatedly pulling the rubber sleeve 42 to rapidly mix the first mixture and the second mixture;

3) the second paraffin plate 30 is melted by raising the temperature, and the first mixture, the second mixture, and the third mixture are mixed by repeatedly pulling the rubber sleeve 42.

EXAMPLE seven

A kit using method comprises the kit as described in the fifth embodiment, and the specific steps are as follows:

1) separating the end cap 44 from the fixing ring 41;

2) rotating the rotating part 431 to make the end of the telescopic rod 434 abut against the tip of the paraffin plate, directly puncture the tip of the first paraffin plate 20 or heat the end of the telescopic rod 434 through a constant temperature heating device, and when the temperature of the end reaches the melting temperature of the paraffin plate, the paraffin is melted to form a hole;

the first mixture and the second mixture are rapidly mixed and homogenized by repeatedly pulling the rubber sleeve 42;

3) repeating the step 2) to form a hole at the tip of the second paraffin sheet 30; the first mixture, the second mixture, and the third mixture are mixed by repeatedly pulling the gum cover 42.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preservation method of an RNA nucleic acid detection reagent comprises mixing reverse transcriptase, 50% glycerol and an RNA inhibitor to prepare a first mixture;

2. The preservation method for the RNA nucleic acid detection reagent according to claim 1, wherein the melting point temperature of the paraffin section between the first mixture and the second mixture is 36 to 38 ℃;

3. A kit comprises a tube body, and is characterized by further comprising a first paraffin sheet and a second paraffin sheet;

4. The kit of claim 3, further comprising a closure assembly comprising a securing ring, a gum cover, and an end cap;

5. The kit of claim 4, wherein the closure assembly further comprises an adjustment assembly;

6. The reagent cartridge according to claim 5, wherein the rotation part center, the connection shaft, the sleeve inner wall, the end of the telescopic rod inside the sleeve, and the telescopic rod inner core and the end are made of metal, and the end of the telescopic rod can be heated up rapidly when the rotation part center is heated.

7. The kit of any one of claims 4 to 6, wherein the paraffin wax flakes are funnel-shaped or cone-shaped, with the tip closed and the tip facing the open end of the tube.

8. The kit according to claim 7, wherein the open end edge of the paraffin plate is provided with a circular paraffin ring having a symmetrical longitudinal section;

9. A kit using method, which is characterized by comprising the kit of claim 4 and comprising the following specific steps:

1) separating the end cover from the fixing ring;

10. A method for using the kit, which comprises the kit of claim 8, and comprises the following steps:

1) separating the end cover from the fixing ring;

3) repeating the step 2) to form a hole at the tip of the second paraffin plate; and repeatedly pulling the rubber sleeve to uniformly mix the first mixture, the second mixture and the third mixture.