阅读说明：本技术 磁珠-核酸复合体洗涤液及应用 (Magnetic bead-nucleic acid complex washing solution and application ) 是由 黄美莹 毕峻龙 刘晓东 李滨 黎明 于 2021-03-24 设计创作，主要内容包括：本发明涉及体外诊断核酸检测领域,提供了一种新的磁珠-核酸复合体洗涤液及应用。本洗涤液由粘度介于1000～50000cs之间的硅油构成。本应用包括将粘度介于1000～50000cs之间的硅油作为磁珠法全自动提取核酸模板的洗涤液。上述硅油作为洗涤液,能够有效的分隔裂解液和扩增液,避免裂解液下渗。而且,不会发生磁珠滞留,能够解决磁珠临界损失问题,避免影响传送到扩增液的核酸模板数量。(The invention relates to the field of in-vitro diagnosis nucleic acid detection, and provides a novel magnetic bead-nucleic acid complex washing solution and application thereof. The cleaning solution is composed of silicone oil with the viscosity of 1000-50000 cs. The application comprises the step of taking silicone oil with the viscosity of 1000-50000 cs as washing liquid for full-automatically extracting the nucleic acid template by a magnetic bead method. The silicone oil is used as a washing liquid, so that the lysis solution and the amplification solution can be effectively separated, and the lysis solution is prevented from leaking downwards. Moreover, the magnetic bead retention can not occur, the problem of critical loss of the magnetic bead can be solved, and the influence on the number of the nucleic acid templates conveyed to the amplification solution can be avoided.)

1. A magnetic bead-nucleic acid complex washing solution is characterized in that the magnetic bead-nucleic acid complex washing solution is composed of silicone oil with the viscosity of 1000-50000 cs.

2. The washing solution for magnetic bead-nucleic acid complex according to claim 1, wherein the viscosity of silicone oil is 3000 to 30000 cs.

3. The washing solution for magnetic bead-nucleic acid complex according to claim 1, wherein the viscosity of the silicone oil is 5000 to 20000 cs.

4. The application of the silicone oil with the viscosity of 1000-50000 cs as the washing liquid for fully automatically extracting the nucleic acid template by a magnetic bead method.

5. The use according to claim 4, wherein the silicone oil is filled between the lysis solution and the amplification solution in the test tube.

6. Use according to claim 5, wherein the silicone oil has a depth of between 30 and 60 mm.

Technical Field

The invention relates to the field of in-vitro diagnosis nucleic acid detection, in particular to a novel magnetic bead-nucleic acid complex washing solution and application thereof.

Background

The magnetic bead method is an automatic nucleic acid extraction method used for nucleic acid detection. The magnetic bead method is characterized in that the magnetic attraction effect of an external magnet and magnetic beads in a test tube is utilized, the external magnet drives the magnetic beads to move in the test tube, the magnetic bead-nucleic acid complex moves from lysis solution to washing solution and then from the washing solution to amplification solution, and therefore the automatic extraction of a nucleic acid template is achieved.

In the magnetic bead method for automatically extracting nucleic acid, the washing solution not only removes the lysis solution and the sample solution remained on the magnetic bead-nucleic acid complex, but also has the function of separating the lysis solution from the amplification solution. The existing magnetic bead method for automatically extracting nucleic acid has the following problems: 1. the problem of lysate infiltration is easy to occur; 2. the magnetic beads are easy to remain on the critical surface of the two liquid components, namely, the problem of critical loss of the magnetic beads exists.

Disclosure of Invention

The present invention is directed to a magnetic bead-nucleic acid complex washing solution and applications thereof, which at least to some extent solve the above-mentioned problems of the related art.

The inventor researches and finds that by using silicone oil with proper viscosity as a washing solution in the automatic nucleic acid extraction by a magnetic bead method, a lysis solution and an amplification solution can be effectively separated, and the problem of critical loss of magnetic beads can be solved, so that the application of the patent is provided. The technical scheme adopted by the invention is as follows:

a washing solution for magnetic bead-nucleic acid complex is composed of silicone oil with viscosity of 1000-50000 cs. The viscosity of the silicone oil can be any value between 1000-50000 cs, such as 1000cs, 2000cs, 2500cs, 3000cs, 4000cs, 5500cs, 6000cs, 7000cs, 8000cs, 9000cs, 10000cs, 12000cs, 13000cs, 15000cs, 16000cs, 18000cs, 20000cs, 21000cs, 23000cs, 25000cs, 28000cs, 30000cs, 33000cs, 35000cs, 37000cs, 40000cs, 43000cs, 45000cs, 48000cs, 49000cs, 50000 cs.

The viscosity of the silicone oil is preferably 3000-30000 cs.

The viscosity of the silicone oil is preferably 5000-20000 cs.

The invention also provides application of the silicone oil with the viscosity of 1000-50000 cs as washing liquid for full-automatic extraction of the nucleic acid template by a magnetic bead method.

In the above application, the silicone oil is filled between the lysis solution and the amplification solution in the test tube.

In the application, the depth of the silicone oil is preferably between 30 and 60 mm.

Compared with the prior art, the invention has at least the following beneficial effects:

experiments prove that the silicone oil in the viscosity range is used as a washing liquid, so that the lysis solution and the amplification solution can be effectively separated, and the lysis solution is prevented from leaking downwards. Moreover, the magnetic bead retention can not occur, the problem of critical loss of the magnetic bead can be solved, and the influence on the number of the nucleic acid templates conveyed to the amplification solution can be avoided.

Drawings

FIG. 1 is a diagram showing the effect of separation by a conventional washing liquid;

FIG. 2 is a diagram showing the separation effect of a silicone oil washing solution;

FIG. 3 is a graph showing the amplification curve using a conventional washing solution;

FIG. 4 is a graph showing the amplification curve using silicone oil as the washing solution;

FIGS. 5 to 7 are graphs showing amplification curves using silicone oils of different viscosities as washing solutions.

Detailed Description

The invention is further illustrated with reference to the following figures and examples.

The first embodiment is as follows: magnetic bead-nucleic acid complex washing solution

The magnetic bead-nucleic acid complex washing solution is composed of silicone oil with the viscosity of 1000-50000 cs. The viscosity of the silicone oil can be any value between 1000-50000 cs, such as 1000cs, 2000cs, 2500cs, 3000cs, 4000cs, 5500cs, 6000cs, 7000cs, 8000cs, 9000cs, 10000cs, 12000cs, 13000cs, 15000cs, 16000cs, 18000cs, 20000cs, 21000cs, 23000cs, 25000cs, 28000cs, 30000cs, 33000cs, 35000cs, 37000cs, 40000cs, 43000cs, 45000cs, 48000cs, 49000cs, 50000 cs. The viscosity of the silicone oil is preferably 3000-30000 cs. The viscosity of the silicone oil is preferably 5000-20000 cs.

Wherein the viscosity of the silicone oil has an influence on the washing effect and the partitioning effect. The specific effects are as follows: when the viscosity is too high, the magnetic beads are retained in the silicone oil, the quantity of the nucleic acid template transmitted to the amplification solution is influenced, the amplification effect is poor, and the result is unstable; when the viscosity is too low, the silicone oil cannot stay in the test tube for a long time, the silicone oil moves upwards, and the lysis solution infiltrates into the amplification solution to denature and inactivate the amplification enzyme.

Example two:

the application of the silicone oil as the washing liquid for full-automatic extraction of the nucleic acid template by a magnetic bead method. The viscosity of the silicone oil can be any value between 1000-50000 cs, such as 1000cs, 2000cs, 2500cs, 3000cs, 4000cs, 5500cs, 6000cs, 7000cs, 8000cs, 9000cs, 10000cs, 12000cs, 13000cs, 15000cs, 16000cs, 18000cs, 20000cs, 21000cs, 23000cs, 25000cs, 28000cs, 30000cs, 33000cs, 35000cs, 37000cs, 40000cs, 43000cs, 45000cs, 48000cs, 49000cs, 50000 cs. The viscosity of the silicone oil is preferably 3000-30000 cs. The viscosity of the silicone oil is preferably 5000-20000 cs.

In the application, the silicone oil is filled between the lysis solution and the amplification solution in the test tube, and the silicone oil is used as a washing solution to remove the residual lysis solution and sample solution of the magnetic bead-nucleic acid complex.

In the application, the depth of the silicone oil is preferably between 30 and 60 mm.

Example three: comparative experiment for washing effect of conventional washing liquid and silicone oil washing liquid

Routine wash liquor experiments: the lysis solution and the washing solution adopt a full-scale gold EC601 universal kit, the amplification solution adopts MD027 amplification solution of Fenpeng company, and the washing effect is shown in figure 1.

Experiment of silicon oil washing solution:

the lysis solution adopts a full-scale gold EC601 universal kit, the amplification solution adopts MD027 amplification solution of Fenpeng company, the washing solution adopts silicone oil with the viscosity of 15000cs, and the washing effect is shown in figure 2.

The lysis solution adopts a full-scale gold EC601 universal kit, the amplification solution adopts MD027 amplification solution of Fengpo company, silicone oil with the viscosity of 5000cs, 8000cs, 12000cs, 20000cs, 30000cs and 40000cs is respectively used as washing solution, and the washing effect is shown in figure 2.

In the test tubes of FIGS. 1 and 2, the upper layer is a lysis solution, the lower layer is an amplification solution, and the middle layer is a washing solution. As can be seen from FIG. 1, the upper layer of the lysis solution has different degrees of downward permeation, and magnetic beads remain in the bottom amplification solution and on the critical surfaces of the washing solution and the amplification solution. As can be seen from FIG. 2, the lysate did not significantly bleed down, and there were no magnetic beads remaining in the amplification solution and at the two critical surfaces. Therefore, compared with the conventional washing liquid, the silicone oil washing liquid has a good separation effect, and can effectively avoid denaturation and inactivation of the amplification enzyme caused by infiltration of the lysis liquid into the amplification liquid; meanwhile, the magnetic beads are not detained in the silicone oil washing liquid, so that the number of the nucleic acid templates transmitted to the amplification liquid is not influenced, and therefore, the silicone oil washing liquid replaces the traditional nucleic acid washing liquid, the separation of the lysis liquid and the amplification liquid is realized, and the problem of critical loss of the magnetic beads is solved.

Example four: comparative experiment of amplification Effect

Routine wash solution amplification experiments: the lysis solution and the washing solution were all-purpose gold EC601 kit, the amplification solution was MD027 amplification solution from philippine corporation, brucella was used as a comparative sample, and the amplification curve is shown in fig. 3, where the ordinate is fluorescence intensity, the abscissa is cycle number, and ROX is negative reference.

Amplification experiment with silicone oil washing solution: the lysis solution adopts a full-scale gold EC601 universal kit, the amplification solution adopts MD027 amplification solution of Fengpo company, the washing solution adopts silicone oil with the viscosity of 15000cs, Brucella is used as a comparison sample, and an amplification curve is shown in FIG. 4, wherein the ordinate is fluorescence intensity, the abscissa is cycle number, and ROX is negative reference.

As can be seen from comparing FIG. 3 and FIG. 4, the washing effect of the silicone oil on the magnetic bead-nucleic acid complex is better than that of the conventional washing solution in terms of the amplification effect.

Example five: experiment for comparing viscosity of silicone oil washing liquid with amplification effect

The lysis solution adopts a full-scale gold EC601 universal kit, the amplification solution adopts MD027 amplification solution of Fengpo corporation, the washing solution respectively adopts silicone oil with viscosity of 55000cs, silicone oil with viscosity of 15000cs and silicone oil with viscosity of 800cs, brucella is used as a comparison sample, and the amplification curve is shown in corresponding figures 5, 6 and 7, wherein the ordinate is fluorescence intensity, the abscissa is cycle number, and ROX is negative reference.

As is apparent from FIGS. 5, 6 and 7, the poor amplification effect and the unstable amplification result are caused by the use of the high-viscosity silicone oil as the washing solution, and the reason for this is that the high-viscosity silicone oil affects the movement speed of the magnetic beads, and the magnetic beads are retained in the silicone oil layer, so that the nucleic acid template cannot be brought into the amplification solution. The reason why amplification cannot be performed by using a low-viscosity silicone oil as a washing liquid is probably that the low-viscosity silicone oil cannot stay in the reaction tube for a long time, the silicone oil moves upward, and the lysate infiltrates into the amplification liquid to denature and inactivate the amplification enzyme. And the silicone oil with proper viscosity is used as a washing liquid, so that the amplification effect is good.

The present invention has been described in detail with reference to the specific embodiments, and the detailed description is only for the purpose of helping those skilled in the art understand the present invention, and is not to be construed as limiting the scope of the present invention. Various modifications, equivalent changes, etc. made by those skilled in the art under the spirit of the present invention shall be included in the protection scope of the present invention.