METHOD FOR SEPARATING NUCLEIC ACID AMPLIFICATION SYSTEM, AND SEPARATOR

Abstract

Provided are a separator for separating a nucleic acid amplification system, comprising a polyethylene wax, a solid paraffin wax, and a liquid paraffin wax. Also provided is a method for separating a nucleic acid amplification system, comprising using the separator as a separation layer to separate a nucleic acid amplification system within a same container. The separating layer can be broken by means of applying an external force, thereby mixing the nucleic acid amplification system.

Claims

1. A separating agent for a nucleic acid amplification system, comprising, the separating agent comprises polyethylene wax, solid paraffin and liquid paraffin.

2. The separating agent according to claim 1, wherein a mass ratio of the polyethylene wax to the solid paraffin to the liquid paraffin is: (0.02-0.05): (0.6-1.2): (4-10).

3. The separating agent according to claim 2, wherein a mass ratio of the polyethylene wax to the solid paraffin to the liquid paraffin is: (0.03-0.05): (0.6-0.8): (4-10).

4. The separating agent according to claim 1, characterized in that, wherein a molecular weight of the polyethylene wax is 1,000 to 10,000.

5. The separating agent according to claim 1, characterized in that, wherein a number of carbon atoms of the solid paraffin is 9 to 30.

6. The separating agent according to claim 1, wherein a number of carbon atoms of the liquid paraffin is 9 to 13.

7. A method for separating a nucleic acid amplification system, comprising, the separating agent of claim 1 is utilized as a separating layer to separate the nucleic acid amplification system in a same container; and the separating layer can be ruptured by applying an external force, so as to realize mixing of the nucleic acid amplification system.

8. The method according to claim 7, wherein the external force is a centrifugal force.

9. The method according to claim 7, wherein the applying the external force is to centrifuge for 5 to 60 seconds under a condition of a centrifugal force not less than 5,000 g.

10. The method according to claim 9, wherein the applying the external force is to centrifuge for 10 to 30 seconds at 5,000 g to 10,000 g, and preferably, the applying the external force is to centrifuge for 20 seconds at 5,000 g.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0033] FIG. 1 is a schematic diagram of separation of PCR reagents in a single-tube.

[0034] FIG. 2 shows PCR results, in which curves without asterisks are for N gene amplification; and curves with asterisks are for 1 ab gene amplification.

DESCRIPTION OF EMBODIMENTS

[0035] The present invention is further illustrated below in combination with specific embodiments, but examples do not limit the present invention in any form. Unless otherwise specified, raw material reagents used in the examples of the present invention are conventionally purchased raw material reagents.

Example 1 Different Formulas of Paraffin Separating Agents

[0036] This example provides a series of different formulas of paraffin separating agents, as shown in Table 1 and Table 2 for details.

TABLE-US-00001 TABLE 1 Mixed paraffin separating agents with different ratios Formulas of Polyethylene Solid Liquid separating agents wax (g) paraffin (g) paraffin (ml) 1 0.02 0.6 4 2 0.02 0.8 6 3 0.02 1.0 8 4 0.02 1.2 10 5 0.03 0.6 6 6 0.03 0.8 4 7 0.03 1.0 10 8 0.03 1.2 8 9 0.04 0.6 8 10 0.04 0.8 10 11 0.04 1.0 4 12 0.04 1.2 6 13 0.05 0.6 10 14 0.05 0.8 8 15 0.05 1.0 6 16 0.05 1.2 4 17 0.01 0.8 4 18 0.06 0.6 10 19 0.03 0.5 4 20 0.05 1.4 10 21 0.03 0.8 2 22 0.05 0.6 12

[0037] In Table 1, the polyethylene wax is polyethylene wax with a molecular weight of 4,000 to 6,000; the solid paraffin is solid paraffin with the carbon atom number of 20 to 25; and the liquid paraffin is liquid paraffin with the carbon atom number of 9 to 11.

TABLE-US-00002 TABLE 2 Mixed paraffin separating agents with different raw materials Formulas of separating Polyethylene Solid Liquid agents wax (0.05 g) paraffin (0.6 g) paraffin (10 mL) 23 The molecular weight The number of The number of is 1,000 to 4,000 carbon atoms is carbon atoms is 20 to 25 9 to 11 24 The molecular weight The number of The number of is 6,000 to 10,000 carbon atoms is carbon atoms is 20 to 25 9 to 11 25 The molecular weight The number of The number of is 4,000 to 6,000 carbon atoms is carbon atoms is 9 to 20 9 to 11 26 The molecular weight The number of The number of is 4,000 to 6,000 carbon atoms is carbon atoms is 25 to 30 9 to 11 27 The molecular weight The number of The number of is 4,000 to 6,000 carbon atoms is carbon atoms is 20 to 25 11 to 13

Example 2 Performance Test of Separating PCR Reagents by Different Formulas of Paraffin Separating Agents

[0038] 1. Separating Method

[0039] A schematic diagram of separation of single-tube PCR reagents is shown in FIG. 1. Specific steps are as follows:

[0040] (1) a PCR 8-tube strip was used, and 15 uL of a PCR buffer solution (containing primers, probes and dNTP) was added to a bottom of the PCR 8-tube strip;

[0041] (2) 27 formulas of paraffin separating agents in Example 1 were used and heated to 100° C. for melting, then 10 uL of paraffin separating agents was added to each tube, and paraffin floated on a surface of the buffer solution and was solidified; and

[0042] (3) a small amount of bromophenol blue was added into an enzyme solution until blue color appeared, 5 uL of the enzyme solution was added to the solidified paraffin layer, the tube was capped, and preparation of the single-tube reagents was completed.

[0043] 2. Stability Test

[0044] (1) The above prepared 27 formulas of single-tube reagents were vortexed for 10 seconds at 3,000 rpm to simulate a transportation process, and the leakage situation was inspected;

[0045] (2) The above prepared 27 formulas of single-tube reagents were put into a centrifuge and centrifuged for 20 seconds with a centrifugal force of 5,000 g, and the leakage situation was inspected; and

[0046] (3) The above prepared 27 formulas of single-tube reagents were placed into a thermostatic metal bath and heated for 2 minutes at 55° C., and the melting situation was inspected, wherein complete melting is considered when a paraffin layer is completely transparent.

[0047] 2. Test Results

[0048] Leakage and melting results are shown in Table 3.

TABLE-US-00003 TABLE 3 Test indexes of different formulas of Oscillation Centrifuging Heating and separating agents leakage leakage melting at 55° C. 1 − + − 2 − + − 3 − + − 4 − + − 5 − + + 6 − + + 7 − + − 8 − + − 9 − + + 10 − + + 11 − + − 12 − + − 13 − + + 14 − + + 15 − + − 16 − + − 17 + + + 18 + + + 19 + + + 20 + + + 21 + + − 22 + + − 23 − − + 24 − + − 25 − − + 26 − + − 27 − − + “+” means leakage or melting, and “−” means no leakage or melting.

[0049] The test results in Table 3 demonstrates that separating agents 1 to 16 did not result leak after severe vortexing, indicating that they can endure oscillation during transportation without rupture; moreover, after centrifuging for 20 seconds at 5,000 g, a separating layer could be ruptured to mix upper and lower layers of solutions, indicating that even mixing can be facilitated during use; in addition, separating agents 5, 6, 9, 10, 13 and 14 could be completely melted after heating for 2 minutes at 55° C., and could be directly used for nucleic acid amplification procedures at an initial temperature of 55° C. and below. Separating agents 1, 2, 3, 4, 7, 8, 11, 12, 15 and 16 cannot be completely melted after heating for 2 minutes at 55° C., but they can be rapidly and completely melted when the temperature reaches 60° C. They can also be suitable for nucleic acid amplification procedures at other initial temperatures and reagent separation of the nucleic acid amplification system. In summary, the melting temperatures of different separating layers are slightly different, and therefore, applicable nucleic acid amplification procedures are also different.

[0050] In addition, although separating agents 17 to 22 could be ruptured after centrifuged for 20 seconds at 5,000 g to mix the upper and lower layers of solutions, leaking situation was observed after vortexing experiment, and therefore, these agents are not suitable for long-distance transportation.

[0051] Separating agents 23, 25 and 27 did not leak after severe vortexing, and were not ruptured completely after centrifuged for 20 seconds at 5,000 g, but they could be ruptured completely after centrifuged for 20 seconds under a centrifugal force increased to 6,000 g; separating agents 24 and 26 did not leak after severe vortexing, and could not be melted completely after heated for 2 minutes at 55° C., but they could be rapidly and completely melted after a temperature rose to 60° C., thus, can also satisfy the reagent separation requirements of the nucleic acid amplification system.

Application Example Preparation of PCR Reagents for Rapid Novel Coronavirus Detection by Using Paraffin Separating Agents

[0052] In this Application Example, separating reagents 1, 3, 6, and 13 in Example 1 were used as examples to separate the PCR reagents for detection of novel coronavirus (2019-nCoV).

[0053] 1. Experimental Method

[0054] A novel coronavirus (2019-nCoV) nucleic acid detection kit (a PCR-fluorescent probe method) is manufactured by DAAN GENE, SUN YAT-SEN UNIVERSITY. According to the separating method in Example 2, separating agents 1, 3, 6 and 13 were used to prepare single-tube reagents. The reagents without separating agents were used as a control group, a positive reference template of 106-103 copies/ml was detected, and PCR procedures were as follows.

[0055] 2 minutes at 55° C.; 2 minutes at 95° C.; (30 seconds at 95° C., 30 seconds at 65° C. to read fluorescence)×40 cycles.

[0056] 2. Experimental Results

[0057] FIG. 2 displays the PCR results showing that amplification curves using mixed paraffin separating agent groups 1 and 3 demonstrated significant fluctuations with respect to a baseline during the first 20 cycles, resulting in wrong interpretation of ct values. This can be explained that paraffin was not completely melted at 55° C., affecting penetration of fluorescence. Fluorescence values and ct values of amplification curves of mixed paraffin separating agent groups 6 and 13 were basically the same as those of a non-paraffin group, indicating that separating agents did not affect the performance of PCR and met performance requirements of separation of PCR reagents.

[0058] The inventor further prepared the mixed paraffin separating agent group 1 and 3 into single-tube form and heated them to 60° C. before initiating PCR procedures. Subsequent results also showed that fluorescence values and ct values of amplification curves were basically the same as those of the non-paraffin group, indicating that separating agents did not affect the performance of PCR.

[0059] Obviously, the above examples of the present invention are only instances for clearly illustrating the present invention, rather than limiting the embodiments of the present invention. For those of ordinary skill in the field, changes or modifications in other different forms may also be conducted on the basis of the above illustration. There is no need and cannot be exhaustive of all embodiments here. Any modification, equivalent replacement, improvement, etc., conducted within the spirit and principle of the present invention should be included within the scope of protection claimed by the present invention.