TWI555849B - Composition for biosample treatment and method for nucleic acid amplification using thereof - Google Patents

Description

Composition for biological sample processing and nucleic acid amplification method using same

The present invention relates to a composition for simplifying the processing of biological samples and a method for nucleic acid amplification using the same.

Commercially available reagents for nucleic acid amplification reactions, such as Qiagen, etc., typically contain several solutions that differ in function. For example, solutions for sample processing include proteinase or lysis buffers, as well as solutions for nucleic acid amplification, including polymerases, deoxyribonucleic acids, buffers, and the like.

However, currently commercially available reagents are often carried out in multiple tubes, which increases the probability of sample contamination during operation. Moreover, since the sample is subjected to a variety of solution treatments, the sensitivity of the final result due to background interference caused by the treatment solution cannot be avoided.

Therefore, in order to reduce the risk of contamination in operation, reduce background interference, and simplify the operation steps, there is a need to develop novel reagents.

An embodiment of the present invention provides a composition for biological sample treatment comprising: at least one halocarbon compound, at least one polyether, and at least one surfactant, wherein the halocarbon compound is present in an amount of 1 of the total weight of the composition. ~70% by weight.

Another embodiment of the present invention provides a method for nucleic acid amplification, comprising: adding at least one halogen hydrocarbon compound to at least one biological sample; a polyether and a composition of at least one surfactant, uniformly mixed to form a homogeneous solution; and adding a reagent for nucleic acid amplification reaction to the homogeneous solution to uniformly mix and perform a nucleic acid amplification reaction; wherein The content of the halocarbon compound is from 1 to 70% by weight based on the total weight of the composition.

The specific embodiments of the present invention are described in detail below, but the following embodiments are only used to further disclose the technical content of the present invention, and should not limit the scope of the invention.

Conventional reagents for nucleic acid amplification, such as Qiagen et al, mainly include reagents for sample processing and reagents for nucleic acid amplification. The reagents used for sample processing typically include a proteinase K that decomposes protein impurities, a homogeneous solution that homogenizes tissue samples, a cytolysis solution that causes tissue lysis, a buffer that binds to nucleic acid molecules, and a washing solution (washing) Buffer) and elution buffer. The reagents for nucleic acid amplification usually include a polymerase, a deoxyribonucleic acid (dNTPs), and a buffer for prolonging and amplifying the nucleic acid.

However, according to the operation manual of the commercially available reagent, the operation is carried out in a separate manner, thereby increasing the probability of sample contamination and increasing the operation time. Furthermore, toxic solvents such as phenol and chloroform must be used, which are not friendly to the operator and the environment. Secondly, the conventional nucleic acid separation method is limited to a single type of sample and its purification sample takes time and labor to obtain better purification results, which is disadvantageous for simultaneously operating a plurality of different sample types and a small or large sample volume.

In order to solve the above-mentioned long-standing technical problems, the present inventors provide A composition that is novel and efficient in processing biological samples and a nucleic acid amplification method using the same. According to the present invention, the cytolysis and homogenization of the biological sample can be completed in a single step in a single tube, and the reagent for nucleic acid amplification can be directly added to the same tube, thereby simplifying the operation flow, reducing the risk of contamination and the operation time. According to the present invention, the nucleic acid amplification result with low background interference can be obtained, and the sensitivity can be improved, and the operable sample volume is in the range of about 1 to 30 μl, which is in line with the demand for the increasingly mature nucleic acid amplification reaction.

In particular, an embodiment of the invention provides a composition for biological sample processing comprising: at least one halocarbon compound, at least one polyether, and at least one surfactant.

The halocarbon compound of the present invention may include a fluorocarbon compound, a chlorocarbon compound, a bromine hydrocarbon compound or an iodocarbon compound, and the like, and a perfluorinated hydrocarbon is preferred. Perfluorinated hydrocarbons are widely used in the electronics industry as a coolant for electronic products. However, the inventors of the present invention have learned that perfluorinated hydrocarbons can remove protein interference when developing biological samples, and are chemically stable in a high temperature and low temperature environment and do not remain in the purification process, thereby improving the biological activity. The purity of the nucleic acid isolated in the sample.

The perfluorinated hydrocarbon used in the present invention may include perfluoroalkanes having 1 to 12 carbons, including tetrafluoromethane, hexafluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane. , perfluoroheptane or perfluorooctane, etc.; and perfluorocycloalkanes having 3 to 12 carbon atoms, such as perfluorocyclopropane, perfluorocyclobutane, perfluorocyclopentane, perfluorocyclohexane, Fluorocycloheptane or perfluorocyclooctane, and the like.

The polyether of the present invention may be, for example, paraformaldehyde, polyoxymethylene, polyacetal. (polyacetal), polyethylene glycol, polyethylene oxide, polyoxyethylene, polypropylene glycol, polypropylene oxide, polyoxypropylene, polytetramethylene Polytetramethylene glycol, polytetramethylene ether glycol, polytetrahydrofuran, or a combination thereof, but is not limited thereto.

The surfactant of the present invention may include a surfactant suitable for nucleic acid separation, and is not particularly limited, and specifically, for example, sodium lauryl sulfate, lithium dodecyl sulfate, and polysorbate. Polysorbate (eg Tween or Tween 80), polyethylene glycol (1,1,3,3-tetramethylbutyl) phenyl ether (polyethyleneglycol4-(1,1,3,3-tetramethylbutyl) -phenyl ether) (for example Triton X-100), or a combination thereof.

In the composition for biological sample treatment of the present invention, the content of the halocarbon compound is preferably from 1 to 70% by weight, more preferably from 10 to 60% by weight, even more preferably 20% by weight based on the total weight of the composition. ~50% by weight. The content of the polyether described in the present invention is preferably from 1 to 50% by weight, more preferably from 5 to 45% by weight, still more preferably from 10 to 40% by weight, based on the total weight of the composition. The content of the surfactant according to the present invention is preferably from 0.01 to 5% by weight, more preferably from 0.1 to 4% by weight, still more preferably from 1 to 3% by weight, based on the total weight of the composition.

The composition for biological sample treatment of the present invention may further comprise at least one reagent for nucleic acid amplification reaction. The reagent for the nucleic acid amplification reaction is not particularly limited, and a reagent prepared by the laboratory itself may be used, or a commercially available reagent may be used. Test for nucleic acid amplification reaction according to the present invention The agent may specifically comprise, for example, a polymerase, deoxyribonucleic acid, a buffer, or a combination thereof.

The ratio of the composition for biological sample treatment of the present invention to the reagent for nucleic acid amplification reaction is not particularly limited. However, in order to obtain a low background interference and high sensitivity, the ratio of the composition for biological sample treatment of the present invention to the reagent for nucleic acid amplification reaction is preferably 1:1 to 1000, more preferably 1:1. ~500, and even better 1:1~200.

The nucleic acid amplification reaction of the present invention may include all current reactions capable of performing nucleic acid amplification, such as, for example, polymerase chain reaction (PCR), real-time polymerase chain reaction (real time-PCR), quantitative real-time polymerase chain reaction ( Real-time quantitative PCR), multiplex PCR, quantitative multiplex quantitative PCR, reverse transcription polymerase chain reaction (RT-PCR), or quantitative reverse transcription polymerase chain reaction ( qRT-PCR).

The biological sample to be treated by the composition of the present invention is also not particularly limited and may include, for example, cells, tissues, blood, serum, urine, amniotic fluid, lymph, saliva, feces, hair, nails or a combination of these.

The nucleic acids of the invention may, for example, be single-stranded nucleic acids, such as RNA; double-stranded nucleic acids, such as DNA; or fragments of such nucleic acids.

By the composition for biological sample processing provided by the present invention, homogenization and cytolysis of the biological sample can be completed in a single tube and in a single step to facilitate subsequent processing of the nucleic acid in the biological sample.

Accordingly, another embodiment of the present invention provides a novel method of nucleic acid amplification comprising the steps of:

Adding a composition containing at least one halogen hydrocarbon compound, at least one polyether, and at least one surfactant to a biological sample, uniformly mixing to form a homogeneous solution; and adding a nucleic acid amplification reaction reagent to the homogeneous solution The mixture is uniformly mixed to carry out a nucleic acid amplification reaction.

The halocarbon compounds, polyethers and surfactants are as defined above. The combination ratio and pH of the composition comprising the halocarbon compound, the polyether and the surfactant are also as defined above.

According to the nucleic acid amplification method of the present invention, the processing time of the biological sample can be simplified, and the background interference caused by the treatment solution can be reduced, so that the nucleic acid molecule of high purity can be efficiently obtained after the amplification reaction.

The reagent for nucleic acid amplification reaction according to the present invention is not particularly limited, and a reagent prepared by the laboratory itself may be used, or a commercially available reagent may be used. The reagent for nucleic acid amplification reaction according to the present invention may specifically comprise, for example, a polymerase, deoxyribonucleic acid, a buffer or a combination thereof.

The compounding ratio of the composition containing a halogen hydrocarbon compound, a polyether, and a surfactant according to the present invention to the reagent for nucleic acid amplification reaction is not particularly limited. However, in order to obtain low background interference and high sensitivity, the formulation ratio of the composition containing the halogen hydrocarbon compound, the polyether and the surfactant to the nucleic acid amplification reaction is preferably 1:1 to 1000, more preferably It is 1:1~500, and even better it is 1:1~200.

The nucleic acid amplification reaction of the present invention may include all current reactions capable of performing nucleic acid amplification, such as, for example, polymerase chain reaction (PCR), real-time polymerase chain reaction (real time-PCR), quantitative real-time polymerase chain reaction ( Real-time Quantitative PCR), multiplex PCR, quantitative multiplex quantitative PCR, reverse transcription polymerase chain reaction (RT-PCR), or quantitative reverse transcription polymerase chain reaction (qRT-PCR) ).

In order to obtain low background interference and high sensitivity, the formulation of the present invention containing at least one halocarbon compound, at least one polyether, and at least one surfactant is preferably formulated at a ratio of 1:1 to the reagent for nucleic acid amplification reaction. ~1000, more preferably 1:1~500, and even more preferably 1:1~200, but not limited to this.

The biological sample of the present invention may include, for example, cells, tissues, blood, serum, urine, amniotic fluid, lymph, saliva, feces, hair, nails or a combination of these.

Further, the nucleic acid of the present invention may include, for example, a single-stranded nucleic acid such as RNA; a double-stranded nucleic acid such as DNA; or a fragment of these nucleic acids.

[Example 1] Comparison with commercially available reagents (1) Purification of nucleic acid using the composition of the present invention (1-1) Preparation of Composition (1)

Take 12.5μl perfluorohexane (Fluorinert ^TM), 10μl of polytetramethylene glycol (polytetramethylene glycol) and 0.1μl of Triton X-100, after uniformly mixed to form a composition (1).

(1-2) Mouse blood amplification reaction

10 μl of mouse blood and 10 μl of serum were added, and the composition (1) prepared above was separately added, uniformly mixed, and allowed to stand for 3 minutes, and then separately reacted with 12.5 μl of a nucleic acid amplification reaction reagent (10 mM (NH ₄ ) ₂ SO ₄ , 10 mM KCl, 2 mM MgSO ₄ , 0.1% Triton X-100 20 mM, Tris-HCl pH 8.8, polymerase enzyme) were uniformly mixed to carry out the following PCR amplification reaction.

(1-3) PCR amplification reaction

A polymerase chain reaction (PCR) amplification reaction under the following conditions was carried out to amplify the target gene GAPDH.

PCR conditions :

Using the above-mentioned nucleic acid amplification reaction reagent and commercially available amplified GAPDH primer pair 1 mM (Fermentas: standard GAPDH primer), amplification was carried out on a PCR amplification machine (ABI 9700) under the following PCR conditions: maintaining temperature 1:95 ° C , 15 minutes; cycle temperature: 95 ° C, 10 seconds; 58 ° C, 30 seconds; and 72 ° C, 30 seconds.

Number of cycles: 40 times.

Maintain the temperature at 2:72 ° C for 7 minutes.

The solution after the above PCR reaction was subjected to electrophoresis (TAE gel, 75 V) to obtain an electrophoretogram shown in Fig. 1.

(2) Purification of nucleic acids using commercially available products (2-1) Amplification reaction of mouse blood

On the other hand, 10 μl of mouse blood and 10 μl of serum were taken according to the Fermentas Mixima® kit and its operating manual. Thereafter, a polymerase chain reaction (PCR) amplification reaction under the following conditions was carried out to amplify the target gene GAPDH.

(2-2) PCR amplification reaction

A polymerase chain reaction (PCR) amplification reaction under the following conditions was carried out to amplify the target gene GAPDH.

PCR conditions:

The Fermentas master mix kit and the commercially available amplified GAPDH primer pair 1 mM (Fermentas Mixima® kit GAPDH standard primer) were amplified on a PCR amplification machine (ABI 9700) according to the following PCR conditions: maintenance temperature 1:95 °C, 15 minutes; cycle temperature: 95 ° C, 10 seconds; 58 ° C, 30 seconds; and 72 ° C, 30 seconds.

Number of cycles: 40 times.

Maintain the temperature at 2:72 ° C for 7 minutes.

The solution after the above PCR reaction was subjected to electrophoresis (TAE gel, 75 V) to obtain an electrophoretogram shown in Fig. 1.

(3) Analysis of the purified DNA sample

According to Figure 1, column L represents the sample strip, columns 1-3 represent the DNA samples after treatment with the Fermentas Mixima® kit, and columns 4-6 indicate the use of Fermentas Mixima. ® sets of DNA samples after treatment of blood, columns 7-9 show DNA samples (Itri PCR) after treatment of serum with composition (1), columns 10-12 show the use of composition (1 A DNA sample (Itri PCR) after treatment of blood.

According to Fig. 1, the amount of DNA sample obtained by PCR amplification of blood and serum treated with the composition (1) is significantly superior to that of a commercially available reagent.

As shown by the results of the present embodiment, the composition of the present invention can complete the cytolysis and homogenization of the biological sample in a single step in a single tube, and can directly add the reagent for nucleic acid amplification in the same tube, thereby simplifying the operation flow. Reduce pollution risks and operating time.

[Examples 2 to 5] Sample volume range processed

The composition (1) prepared in Example 1 was mixed with an equal volume of the sample blood as shown in the following Table 1, and then mixed with the nucleic acid amplification reaction reagent. Thereafter, the primer for amplifying GAPDH was subjected to denaturation at 95 °C on a PCR machine (ABI 9700) by a primer of 1 mM (Fermentas Mixima® kit GAPDH standard primer); a PCR reaction at a temperature of 58 ° C, a cycle number of 40, and amplification of a labeled primer in the blood. DNA sample. The solution after the above PCR reaction was subjected to electrophoresis of 75 V and TAE gel to obtain an electrophoresis pattern shown in Fig. 2 .

In the second panel, column L represents the sample strip, columns 1-4 show the amplified DNA sample of Example 5, columns 5-8 show the amplified DNA sample of Example 4, column 9 In the blank column, columns 10-12 show the amplified DNA samples of Example 3, and columns 13-15 show the amplified DNA samples of Example 2.

As shown by the results of Examples 2 to 5, the composition of the present invention can expand the range of sample volumes for operation, in accordance with the requirements of nucleic acid amplification reactions.

[Example 6] Application to real-time PCR

The composition (1) prepared in Example 1 was mixed with an equal volume of 10 μl of blood of the sample, and then mixed with a nucleic acid amplification reaction reagent. Then, the primers of 18S/GAPDH were amplified to 1 mM (ABI PCR control 18S/GAPDH standard primer) on a PCR machine (ABI 7500) for denaturation at 95 ° C; 58 ° C bonding, 40 cycles of PCR reaction, amplification of blood The DNA/RNA sample of the primer was labeled and the results are shown in Figures 3 and 4.

Figure 3 shows an amplification map of RNA 18S obtained using the ABI standard 18S primer and the carbon needle group. Fig. 4 is a view showing a GAPDH section obtained by using an ABI standard 18S primer and a carbon needle group.

As shown by the results of this example, the composition of the present invention can be applied to the application of real-time PCR, which meets the requirements of nucleic acid amplification reactions.

[Example 7] Application of detecting virus samples

Composition (1) prepared in Example 1 and standard serum sample (dengue) The virus FDA standard was mixed in an equal volume, and after standing at room temperature for 3 minutes, the nucleic acid amplification reaction reagent was added and mixed. Thereafter, the dengue fever standard primer pair (Disease Control Agency (FDA) standard primer) was applied to the PCR machine (ABI 9700) at a temperature of 95 ° C for 15 minutes; 95 ° C, 30 seconds; 60 ° C, 30 Seconds; RT-PCR at 72 ° C, 30 seconds, PCR number of cycles: 40, amplification of RNA samples of labeled primers in blood. The solution after the above PCR reaction was subjected to electrophoresis of 75 V and TAE gel to obtain an electrophoretogram shown in Fig. 5.

Column L in Figure 5 indicates a sample strip, columns 1-6 indicate DNA samples treated with composition (1), and columns 7-12 indicate DNA samples treated with Qiagen RT-PCR buffer. As shown in Fig. 5, a sample which was treated with the composition of the present example was subjected to an RT-PCR amplification process to obtain a band which was clear on the electropherogram.

As shown by the results of this example, the compositions of the present invention can be used to manipulate viral pathogens in accordance with the requirements of nucleic acid amplification reactions.

[Example 8] Application of detecting tissue samples (1) Amplification of tissue samples using the composition of the present invention (1-1) Amplification of mouse tissues

0.1 mg of frozen mouse spleen tissue was taken, 12.5 μl of the composition (1) prepared in the above Example 1 was added, uniformly mixed, and allowed to stand for 3 minutes, and then reacted with 12.5 μl of nucleic acid amplification reaction reagent (10 mM (NH ₄ )). ₂ SO ₄ , 10 mM KCl, 2 mM MgSO ₄ , 0.1% Triton X-100 20 mM, Tris-HCl pH 8.8, polymerase enzyme) were uniformly mixed to carry out the following PCR amplification reaction.

(1-2) PCR amplification reaction

A polymerase chain reaction (PCR) amplification reaction under the following conditions was carried out to amplify the target gene GAPDH.

PCR conditions:

Using a nucleic acid amplification reaction reagent and a commercially available amplified GAPDH primer pair 1 mM (RefSeq: NM_008084.2), amplification was carried out on a PCR amplification machine (ABI 9700) under the following PCR conditions: maintaining a temperature of 1:95 ° C, 15 minutes; cycle temperature: 95 ° C, 10 seconds; 60 ° C, 30 seconds; and 72 ° C, 30 seconds.

Number of cycles: 40 times.

Maintain the temperature at 2:72 ° C for 7 minutes.

The solution after the above PCR reaction was subjected to electrophoresis (TAE gel, 75 V) to obtain an electrophoretogram shown in Fig. 6.

(2) Amplification of tissue samples using commercially available products (2-1) Amplification of mouse tissues

RNA was purified from Qiagen RNAeasy kit using 0.1 mg of frozen mouse spleen tissue and processed according to Qiagen one-step RT-PCR kit and its operating manual. Thereafter, a polymerase chain reaction (PCR) amplification reaction under the following conditions was carried out to amplify the target GAPDH (RefSeq: NM_008084.2).

(2-2) PCR amplification reaction

A polymerase chain reaction (PCR) amplification reaction under the following conditions was carried out to amplify the target gene GAPDH.

PCR conditions:

The Qiagen one-step RT-PCR kit and GAPDH (RefSeq: NM_008084.2) were used for amplification on a PCR amplification machine (ABI 9700) according to the following PCR conditions: maintenance temperature 1:95 ° C, 15 minutes; cycle Temperature: 95 ° C, 10 seconds; 60 ° C, 30 seconds; and 72 ° C, 30 seconds.

Number of cycles: 40 times.

Maintain the temperature at 2:72 ° C for 7 minutes.

The solution after the above PCR reaction was subjected to electrophoresis (TAE gel, 75 V) to obtain an electrophoretogram shown in Fig. 6.

In Fig. 6, column L indicates a sample strip, columns 1-4 indicate RNA 18S in mouse spleen tissue treated with composition (1) of the present embodiment, and columns 5-9 indicate Qiagen GAPDH in mouse spleen tissue treated with RT-PCR buffer, columns 10-17 show GAPDH in mouse spleen tissue treated with composition (1) of this example.

As shown by the results of this example, the composition of the present invention can be expanded to be applied to biological tissue samples in accordance with the requirements of nucleic acid amplification reactions.

[Example 9] Application in multiplex PCR (multiplex PCR)

5 μl of the composition (1) of Example 1 was mixed with the sample blood in an equal volume, and then mixed with the nucleic acid amplification reaction reagent. Thereafter, 1 mM (ABI control Primer Beta-actin 4352341E, GAPDH 4308313) was amplified on a PCR machine (ABI 9700) at 95 ° C for 15 minutes using an primer for amplifying GAPDH and β-actin (β-actin); °C, 10 seconds; 60 ° C, 30 seconds; 70 ° C, 30 seconds; 72 ° C, 30 seconds, 35 cycles, amplification of DNA samples of labeled primers in the blood. The solution after the above PCR reaction was subjected to electrophoresis of 75 V and TAE gel to obtain an electrophoretogram shown in Fig. 7. Control samples were ABI HUMAN control DNA 4312660 (10 ^-3 μg) and control serum samples (without DNA).

The obtained PCR-amplified sample was subjected to electrophoresis (electrophoresis: TAE, electrophoresis: 75 V) to obtain an electropherogram shown in Fig. 7. Column L in Figure 7 indicates the sample strip, column 1 indicates GADPH, column 2 indicates β-actin-1, column 3 indicates blank column, and column 4 indicates β-actin- 2. Columns 5-6 indicate blank columns.

As shown by the results of this example, the compositions of the present invention can be applied to multiple gene amplifications in accordance with the requirements of nucleic acid amplification reactions.

[Example 10] Application to plant tissues

1 mg of Semen Coicis, Vigna umbellate, Adenanthera pavonina , rice, and brown rice were mixed with 25 μl of the composition (1) of Example 1, respectively, and then The nucleic acid amplification reaction reagent is added and mixed. Thereafter, the amplification (NCBI plant identification standard primer sequence (EF1, E1F, 18S, UBQ, T, ACT2, AC11, TUA) was performed at 95 °C on a PCR machine (ABI 9700); 60 °C bonding, number of cycles: 40 The PCR reaction amplifies the DNA sample of the labeled primer. The solution after the above PCR reaction is subjected to electrophoresis of 75 V and TAE gel to obtain an electrophoresis pattern shown in Fig. 8. In Fig. 8, the column L indicates the sample strip ( Ladder), columns 1-5 indicate the gene UBQ5 in five rice, rice bean, red bean, white rice and brown rice. Columns 6-10 sequentially indicate five rice, rice bean, red bean, white rice and brown rice. Gene UBQ10, columns 11-15 sequentially represent 25S rRNA in five rice, rice bean, red bean, white rice and brown rice, and columns 16-20 sequentially indicate five rice, rice bean, red bean, white rice and brown rice. 18S rRNA, columns 21-24 sequentially represent the gene UBC in five rice, rice beans, red beans, and white rice.

As shown by the results of this example, the composition of the present invention can be applied to plant tissue gene amplification, which meets the requirements of nucleic acid amplification reactions.

[Example 11] A wide range of composition ratio

The nucleic acid amplification reaction of (1-2) and (1-3) of the above Example 1 was repeated according to the volume preparation compositions (2) to (10) shown in Table 2 below.

As shown in the results of Table 2, the compositions of the present invention formulated in different ratios can also meet the requirements of nucleic acid amplification reactions.

While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

Fig. 1 is an electrophoretogram showing a DNA sample obtained by PCR amplification after processing a biological sample using the composition of one embodiment of the present invention and a commercially available reagent.

Figure 2 is an electropherogram showing the DNA sample obtained by PCR amplification after treatment of a plurality of sample volumes using the composition of one embodiment of the present invention.

Figure 3 is a graph showing the amplification of 18S RNA obtained by real-time PCR amplification of a blood sample using the composition of one embodiment of the present invention.

Figure 4 is a graph showing the amplification of the GAPDH interval obtained by real-time PCR amplification after treatment of a blood sample using the composition of one embodiment of the present invention.

Fig. 5 is a view showing an electrophoretogram of a DNA sample obtained by RT-PCR amplification of a dengue virus sample after treatment with a composition of an embodiment of the present invention and a commercially available reagent.

Fig. 6 is an electrophoretogram showing 18S RNA and GAPDH obtained by RT-PCR amplification of blood and spleen tissue samples using a composition according to an embodiment of the present invention and a commercially available reagent.

Fig. 7 is an electrophoretogram showing the GAPDH, β-actin 01 gene, and β-actin 02 gene obtained by multiplex PCR amplification of a serum sample using a composition according to an embodiment of the present invention and a commercially available reagent.

Fig. 8 is an electrophoresis diagram showing various genes obtained by PCR amplification of a plant tissue sample after the composition of one embodiment of the present invention.

Claims (27)

A composition for biological sample treatment comprising: at least one halocarbon compound, at least one polyether, and at least one surfactant, wherein the halocarbon compound is present in an amount from 1 to 70% by weight based on the total weight of the composition. The composition for biological sample treatment of claim 1, wherein the halocarbon compound comprises a perfluorinated hydrocarbon. The composition for biological sample treatment according to claim 2, wherein the perfluorinated hydrocarbon comprises tetrafluoromethane, hexafluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, all Fluorohexane, perfluoroheptane, perfluorooctane or a combination thereof. The composition for biological sample treatment according to claim 1, wherein the polyether comprises paraformaldehyde, polyoxymethylene, polyacetal, polyethylene glycol, Polyethylene oxide, polyoxyethylene, polypropylene glycol, polypropylene oxide, polyoxypropylene, polytetramethylene glycol, polytetramethyl Polytetramethylene ether glycol, polytetrahydrofuran, or a combination thereof. The composition for biological sample treatment according to claim 1, wherein the surfactant comprises sodium lauryl sulfate, lithium dodecyl sulfate, and polysorbate. Alcohol ester, polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether, Or a combination of the above. The composition for biological sample treatment according to claim 1, wherein the polyether is contained in an amount of from 1 to 50% by weight based on the total weight of the composition. The composition for biological sample treatment according to claim 1, wherein the surfactant is contained in an amount of from 0.01 to 5% by weight based on the total weight of the composition. A composition for biological sample treatment as described in claim 1, wherein the composition has a pH of from 7 to 14. The composition for biological sample treatment according to claim 1, wherein the composition further comprises at least one reagent for nucleic acid amplification reaction. The composition for biological sample processing according to claim 9, wherein the reagent for nucleic acid amplification reaction comprises a polymerase, deoxyribonucleic acid, a buffer, or a combination thereof. The composition for biological sample treatment according to claim 9, wherein the ratio of the composition to the reagent for nucleic acid amplification reaction is 1:1 to 1000. The composition for biological sample processing according to claim 9, wherein the nucleic acid amplification reaction comprises polymerase chain reaction (PCR), real-time polymerase chain reaction (real time-PCR), quantitative instant polymerase Real-time quantitative PCR, multiplex PCR, quantitative multiplex quantitative PCR, reverse transcription polymerase chain reaction (RT-PCR), or quantitative reverse transcription polymerization Enzyme chain reaction (qRT-PCR). The composition for biological sample treatment according to claim 1, wherein the biological sample comprises cells, tissues, blood, serum, urine, amniotic fluid, lymph, saliva, feces, hair, nails or the like. combination. The composition for biological sample processing as described in claim 1, wherein the nucleic acid comprises a single-stranded nucleic acid, a double-stranded nucleic acid, a nucleic acid fragment, or a combination of these. A method for nucleic acid amplification comprising the steps of: adding a composition comprising at least one halocarbon compound, at least one polyether, and at least one surfactant to a biological sample, uniformly mixing to form a homogeneous solution; The reagent for nucleic acid amplification reaction is added to the homogeneous solution, and uniformly mixed to carry out a nucleic acid amplification reaction; wherein the content of the halogen hydrocarbon compound is 1 to 70% by weight based on the total weight of the composition. The method of nucleic acid amplification according to claim 15, wherein the halocarbon compound comprises a perfluorinated hydrocarbon. The method of nucleic acid amplification according to claim 16, wherein the perfluorinated hydrocarbon comprises tetrafluoromethane, hexafluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane , perfluoroheptane, perfluorooctane or a combination thereof. The method of nucleic acid amplification according to claim 15, wherein the polyether comprises paraformaldehyde, polyoxymethylene, polyacetal, polyethylene glycol, poly Polyethylene oxide, polyoxyethylene, polypropylene glycol, polypropylene oxide, polyoxypropylene, polytetramethylene glycol, polytetramethyl Polytetramethylene ether glycol, polytetrahydrofuran, or a combination thereof. The method of nucleic acid amplification according to claim 15, wherein the surfactant comprises sodium lauryl sulfate, lithium dodecyl sulfate, polysorbate, Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether, or a combination thereof. The method of nucleic acid amplification according to claim 15, wherein the polyether is contained in an amount of from 1 to 50% by weight based on the total weight of the composition. The method of nucleic acid amplification according to claim 15, wherein the surfactant is present in an amount of from 0.01 to 5% by weight based on the total weight of the composition. The method of nucleic acid amplification according to claim 15, wherein the composition has a pH of 7 to 14. The method of nucleic acid amplification according to claim 15, wherein the nucleic acid amplification reaction reagent comprises a polymerase, a deoxyribonucleic acid, a buffer, or a combination thereof. The method of nucleic acid amplification according to claim 15, wherein the ratio of the composition to the reagent for the nucleic acid amplification reaction is 1:1 to 1000. a method for nucleic acid amplification according to claim 15 of the patent application, The nucleic acid amplification reaction includes polymerase chain reaction (PCR), real time-PCR, real-time quantitative PCR, and multiplex PCR. Quantitative multiplex quantitative PCR, reverse transcription polymerase chain reaction (RT-PCR), or quantitative reverse transcription polymerase chain reaction (qRT-PCR). The method of nucleic acid amplification according to claim 15, wherein the biological sample comprises cells, tissues, blood, serum, urine, amniotic fluid, lymph, saliva, feces, hair, nails or a combination thereof. The method of nucleic acid amplification according to claim 15, wherein the nucleic acid comprises a single-stranded nucleic acid, a double-stranded nucleic acid, a nucleic acid fragment, or a combination thereof.