KR101985432B1 - Method for pretreating urine sample for extracting nucleic acids - Google Patents

Abstract

The present invention relates to a method for pretreatment of a urine sample for nucleic acid extraction, which comprises the steps of pretreating a urine sample, a method of pretreating a urine sample to be used for nucleic acid amplification experiments, . When a urine sample containing cells and other nucleic acids is passed through a membrane (filter) and a solid component that does not dissolve in the urine is filtered through the membrane, the nucleic acid treatment solution is added to the membrane after the filtering step is completed, When the nucleic acid is separated from the cells or the like by dissolving the components filtered by the filter, air pressure is applied to the membrane, and the solution adhered to the membrane is pushed out of the membrane and collected in a clean container to obtain a solution containing the nucleic acid Respectively. Thus, according to the present invention, a nucleic acid can be obtained simply and inexpensively from components contained in urine.

Description

TECHNICAL FIELD The present invention relates to a method for pretreating urine samples,

The present invention relates to a method for pretreatment of a urine sample for nucleic acid extraction, which comprises the steps of pretreating a urine sample, a method of pretreating a urine sample to be used for nucleic acid amplification experiments, .

The urine contains mainly electrolytes and metabolites dissolved in water and water, which are filtered through the kidneys. In addition, there are various kinds of cells derived from the human body such as cells that pass through the urinary system, immune cells, and the like. These cells contain nucleic acids contained in the nucleus and cytoplasm. In addition, there are DNA fragments that are dissolved in water and cell free nucleic acid in the form of mRNA. These nucleic acids are known to provide various kinds of genetic information of the human body when they are used as a template in the nucleic acid amplification assay Therefore, there is a need for a method of effectively treating nucleic acid in the urine in order to utilize it for research and diagnosis of various diseases.

One of the biggest problems in the separation of nucleic acids from the urine is that the concentration of nucleic acids in the urine is too low to be used in experiments. Therefore, the method of isolating nucleic acid from urine, which is mainly used in the present day, mainly uses a method of separating nucleic acid from cells after precipitation of cells existing in urine through ultrafast centrifugation. As another method, there are known kits and methods for separating pure nucleic acids by using multiple columns and centrifugation methods in combination. However, since the known method requires an ultra-high-speed centrifuge or a complicated procedure, it is difficult to carry out the experiment in a place where the medical field or the experimental facility is not fully equipped. Therefore, Development of methods for the separation of nucleic acids in urine of urine is still required.

United States Patent No. 07893228 (registered on February 22, 2011)

It is an object of the present invention to provide a method for pretreating a urine sample which can be used in a nucleic acid amplification test by nucleic acid contained in various urine constituents including cells present in a urine sample through pretreatment of a urine sample.

In order to accomplish the above object, the present invention provides a method for preparing a composition comprising: (1) passing separated urine through a hydrophilic membrane; (2) applying air pressure to the membrane; (3) preparing a pretreatment solution containing NaCl, Tris-HCl and Triton X-100 and adjusted to a final pH of 6 to 9; (4) injecting the prepared pretreatment solution into the membrane to react; And (5) adding air pressure to the membrane to obtain a nucleic acid extraction sample. The present invention also provides a method for pretreating a urine sample for nucleic acid extraction.

The present invention relates to a method for pretreatment of a urine sample for nucleic acid extraction, which comprises the steps of pretreating a urine sample, a method of pretreating a urine sample to be used for nucleic acid amplification experiments, . When a urine sample containing cells and other nucleic acids is passed through a membrane (filter) and a solid component that does not dissolve in the urine is filtered through the membrane, the nucleic acid treatment solution is added to the membrane after the filtering step is completed, When the nucleic acid is separated from the cells or the like by dissolving the components filtered by the filter, air pressure is applied to the membrane, and the solution adhered to the membrane is pushed out of the membrane and collected in a clean container to obtain a solution containing the nucleic acid Respectively. Thus, according to the present invention, a nucleic acid can be obtained simply and inexpensively from components contained in urine.

Fig. 1 shows the result of analysis by electrophoresis after nucleic acid amplification by PCR method. M is a DNA ladder, and an odd numbered lane is a template obtained by treating a membrane with a pretreatment solution, and an even numbered lane indicates a nucleic acid using a conventional extraction method in a urine passing through a membrane. Extracted and used as a template for PCR reaction. 1 and 2 are the same pair of urine samples. All eight urine samples were used repeatedly. Polyvinylidene difluoride (PVDF) membranes were used for lanes 1 to 8, and Nylon (NY) membranes for lanes 9 to 16 were used.
FIG. 2 is a graph showing the results of measurement of the permeability of the final solution obtained by treating 200 μl of the pretreatment solution developed in the present invention for 5 minutes and pneumatic pressure to 5 ml of a 0.45 μm pore size membrane made of PVDF, .
FIG. 3 is a graph showing the results of a comparison between a pretreatment solution developed by the present invention or a Qiagen's buffer for nucleic acid extraction, which is prepared by passing urine samples through a membrane, After air pressure was applied, the solution adhered to the membrane was separated and collected in a clean container, and a certain amount of the solution was used as a template for PCR reaction, amplifying the nucleic acid, and analyzing the reaction product by electrophoresis. Lane 1 is a DNA ladder, lane 1 is a pretreatment solution developed in the present invention, and lane 2 is a Qiagen buffer when 2 μl of the collected solution is used as a template in a urine-permeated membrane. Lane 3 was prepared by treating the membrane of the present invention with the pretreatment solution of the present invention and then using 8 μl of the collected solution as a template in the PCR reaction, In case lane 4 is used as a template in the PCR reaction, 8 ㎕ of the solution collected after treating the membrane with Qiagen buffer, lane 5 is obtained by extracting pure nucleic acid from the cells using the pretreatment solution of the present invention, Lane 6 is a case in which pure nucleic acid is extracted from a cell using Qiagen buffer and the nucleic acid amplification is performed by PCR using the DNA as a template, NC negative contrast And PC is a positive control group.
FIG. 4 is a graph showing the results obtained by passing a urine sample through a membrane and then treating a predetermined amount of a pretreatment solution developed in the present invention or a buffer for nucleic acid extraction of Qiagen to dissolve materials including cells attached to the membrane to liberate the nucleic acid in a free state And shows the reaction when the solution adhered to the membrane is separated and obtained in a clean container by applying air pressure and then a certain amount of the solution is used as a template for LAMP reaction. For the LAMP reaction, nucleic acid amplification is performed using a primer specific for human beta globin gene, and the result is analyzed by electrophoresis. M is a DNA ladder, lane 1 is a pretreatment solution developed by the present invention, and 2 占 퐇 of the collected solution is used as a template for LAMP reaction. , 8 ㎕ of the collected solution was used as a template for LAMP reaction, lane 3 was used as a template for LAMP reaction when 2 ㎕ of the collected solution was treated with Qiagen buffer, and lane 4, when Qiagen buffer was treated with membrane and 8 μl of the collected solution was used as a template for LAMP reaction, lane 5 was obtained by extracting pure nucleic acid from cells using Qiagen buffer, lane 6 is a case where pure nucleic acid is extracted from a cell using the pretreatment solution developed in the present invention and then the DNA is used as a template for LAMP reaction. NC is a negative control, PC is a positive control to be.

Accordingly, the present inventors have made efforts to develop a method capable of easily and easily obtaining a nucleic acid in cells existing in the urine, and as a result, it has been found that a urine sample containing cells and other nucleic acids is passed through a membrane After the filtering step is completed by using the point that the solid component is filtered on the membrane, the nucleic acid treatment solution is added to the membrane, and after reacting for a predetermined time, the component filtered by the filter is dissolved and when the nucleic acid is separated from the cells, A method of extracting a solution containing a nucleic acid was developed by pneumatically applying a solution adhering to a membrane and collecting it in a clean container. By using the above-described method, a treatment method suitable for effectively using nucleic acids in a urine sample containing nucleic acid was confirmed, and the present invention was completed.

(1) passing separated urine through a hydrophilic membrane; (2) applying air pressure to the membrane; (3) preparing a pretreatment solution containing NaCl, Tris-HCl and Triton X-100 and adjusted to a final pH of 6 to 9; (4) injecting the prepared pretreatment solution into the membrane to react; And (5) adding air pressure to the membrane to obtain a nucleic acid extraction sample. The present invention also provides a method for pretreating a urine sample for nucleic acid extraction.

Preferably, the urine may be from 1 to 30 ml, but is not limited thereto.

Preferably, the hydrophilic film may be polyvinylidene difluoride (PVDF) or nylon (NY) film, but is not limited thereto.

More preferably, the hydrophilic membrane may have a pore size of 0.2 to 10 μm and a diameter of 13 to 25 mm, but is not limited thereto.

Preferably, the step of applying air pressure to the membrane may be by injecting 10 to 80 cc of air for 10 to 20 seconds to apply an air pressure of 0.007 to 0.008 cc / mm < 2 > / sec.

Preferably, the step of preparing the pretreatment solution comprises preparing a pretreatment solution containing 40 to 60 mM NaCl, 40 to 60 mM Tris-HCl and 0.01 to 1% Triton X-100 and adjusted to a final pH of 6 to 9 But is not limited thereto.

Preferably, 100 to 600 μl of the pretreatment solution can be injected in the step (4), but the present invention is not limited thereto.

Preferably, the reaction of step (4) may be carried out at room temperature for 5 to 10 minutes, but is not limited thereto.

Preferably, the nucleic acid may be DNA, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to embodiments which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

< Example  1> Pretreatment process

The purpose of the pretreatment process is to keep the amount and quality of the nucleic acid contained in the solution obtained at the final stage sufficient for use in nucleic acid amplification experiments. Factors affecting the quantity and quality of nucleic acids are the conditions under which conditions the experiments are carried out at each step of the preprocessing process.

The preprocessing process according to the present invention is as follows.

(1) The collected urine sample is put into a syringe and pushed through the inlet of a membrane structure in the form of a syringe filter, and the liquid component of the urine is discharged through the membrane to the outlet. Liquid components that pass through the membrane and exit the outlet are discarded.

(2) Solid components, including cells in the urine, can not pass through the membrane and remain on the membrane.

(3) At this time, there is a liquid component remaining in the film in addition to the solid component, which should be removed as much as possible because it may affect the components of the pretreatment solution when the pretreatment solution is diluted. In order to remove the liquid component of the urine that remains in the membrane, air is injected through the injection port by a syringe to push out the liquid component present in the membrane and make the membrane dry properly.

(4) Inject a proper amount of pretreatment solution into the syringe through the injection port.

(5) The pretreatment solution is allowed to react for a certain period of time. The pretreatment solution destroys the solid components remaining on the surface of the membrane so that the nucleic acid is completely separated from the solid component such as cells and is dissolved in the pretreatment solution.

(6) When the reaction is completed, air pressure is applied to the membrane again using a syringe, and the pre-treatment solution present in the membrane is completely pushed out through the outlet to collect as much of the solution as possible for the nucleic acid amplification experiment .

(7) The discharged pretreatment solution is a liquid component containing nucleic acid and collects this solution in a clean container. The collected solution can be directly used for the nucleic acid amplification reaction without any manipulation. The entire process of pretreating the nucleic acid proceeds at room temperature.

< Example  2> Analysis according to amount of urine sample

The amount of urine will determine the degree of permeability through the membrane. Excessive amounts of urine can block the pores of the membrane, increasing the pressure on the membrane and, in severe cases, tearing the membrane so that it can not act to retain the original desired solids content on the membrane.

In the optimal experiment conditions found in the present invention, it is recommended to obtain the most satisfactory results when using 1 to 10 ml of urine, but it is possible to put the urine of up to 30 ml into the usable range.

< Example  3> Analysis according to material and structure of membrane

1. Analysis according to material of membrane

In the first experiment, various types of membranes were used to evaluate the quality of the membranes and the urine was filtered.

Hydrophobic membranes were able to pass through when the urine was pushed through the membrane at high pressure initially. However, the amount of passing was not enough to handle the desired amount of urine. In addition, when the urine passes through the membrane, the pressure applied to the membrane becomes too high and the membrane often tears. Because of these disadvantages, it was judged that the purpose of retaining the cells or solid components contained in the urine on the membrane surface could not be achieved properly, and the hydrophobic membrane was excluded from the first selection process.

The hydrophilic membranes were made from paper made household filters, whatmann filter paper, polyvinylidene difluoride (PVDF) or nylon (NY) membranes.

In the case of household paper filters, the membrane was easily damaged when passing urine. Also, even if the air pressure was applied to the membrane, the moisture remaining in the membrane tended to remain unremoved and was not suitable for the purpose of adding the pretreatment solution.

For the Whatmann filter paper, a grade 1 product with a pore size of 11 μm was used, but the moisture removal was somewhat better and durability was higher than that of a household paper filter, but the case where the solid component such as a cell did not remain on the membrane And were excluded from the second selection process.

In the case of PVDF or NY membrane, durability was excellent and no problems were found in the process of removing moisture from the membrane. When the nucleic acid was extracted using the liquid component passed through the membrane and the solid component remaining on the membrane, and the nucleic acid amplification reaction was used, the components remaining in the membrane were amplified by the nucleic acid amplification reaction to confirm that the membrane functions properly (FIG.

As shown in Figure 1, except for lane 9 and lane 13, all nucleic acid amplification product bands appeared only in the solution treated in the membrane. No products were found in the samples treated with urine passed through the membrane. In the case of lane 9 and lane 13, it was judged that urine samples could not be measured from the beginning because the products were not confirmed in repeated experiments in the untreated urine samples.

2. Analysis according to membrane structure

One of the important factors in the membrane structure is the pore size.

Considering that the largest solid component present in the urine is a cell, the pore size range suitable for the purpose of the present invention is determined to be from 0.2 to 10 μm. Based on this, the PDVF or NY membrane used in the final selection step The pore size was 0.22 μm and 0.45 μm.

To summarize the results of applying PDVF or NY membranes to urine samples, there was no difference in pressure between injecting urine into a syringe at two pore sizes for membranes of the same cross-sectional area (diameter). Compared with the PVDF membranes, the NY membrane showed higher pressure than the PVDF membranes when the pore size and cross - sectional area were the same. Compared with the same pore size and material, the pressure was low when the cross-sectional area was wide.

Another important factor in the membrane structure is the cross-sectional area of the membrane through which the urine passes.

The cross-sectional area of the membrane can be expressed in terms of its diameter because the structure of the membrane is circular. The diameters of the PDVF or NY membranes used in the final experiment were 25 mm and 13 mm, respectively.

The pressure on the 13 mm diameter membrane was higher than that of the 25 mm diameter membrane. When the urine was filtered through the membrane, a considerable amount of solid matter was filtered through the membrane, and the filtered urine showed a clear state even when viewed from the naked eye. This is the result of confirming that the membrane acts properly as a filter. The visual inspection results of the samples obtained after completing all preprocessing steps are shown in FIG.

As shown in FIG. 2, the state of the final obtained sample solution is slightly opaque and has a weak yellow color although the pretreatment solution is in a colorless transparent state. However, unlike normal nucleic acid extraction products, various impurities contained in urine are present to some extent in the final sample. However, considering the results in FIG. 1, it can be seen that there is no problem in using the nucleic acid amplification reaction.

< Example  4> Analysis according to composition and amount of pretreatment solution

1. Analysis by composition of pretreatment solution

The composition of the pretreatment solution used to treat the nucleic acid contained in the urine was composed of 40 to 60 mM NaCl, 40 to 60 mM Tris-HCl, 0.01 to 1% Triton X-100, and the final pH was adjusted to 6 to 9 Respectively. The optimum conditions for the composition were 50 mM NaCl, 50 mM Tris-HCl, 0.1% Triton X-100, and the final pH was adjusted to the composition of the various reagents used to make the optimized composition of the pretreatment solution. Is 7.8.

The concentrations of NaCl and Triton X-100 are important in the concentration of the pretreatment solution. These two concentrations determine the degree to which nucleic acids are separated from solid components such as cells.

The pH was determined to the extent that the final sample did not affect the nucleic acid amplification reaction when used in the nucleic acid amplification reaction.

In the case of the present invention, the concentration of the nucleic acid in the final product solution was measured. As a result, the concentration of the nucleic acid in the nano-drop apparatus could not be measured and the nucleic acid amplification experiment could not be used.

As a result, the concentration and purity of the final nucleic acid solution reached a level suitable for the nucleic acid amplification reaction (Table 1) .

As compared with the case of extracting nucleic acid from Z and N products, which are exclusive products for extracting nucleic acid from the urine samples already existing, there was almost no difference in nucleic acid concentration and 260/280 ratio values were similar. It is judged to be similar. The 260/230 ratio was lower than that of the comparative product. This is because the method developed in the present invention seems to be because there is no separate procedure for removing the protein, but it is considered to be no problem since it does not greatly affect the nucleic acid amplification reaction .

Method ng / ml 260/280 260/230 Company Z 49.2 1.81 1.41 Company N 60.9 1.93 1.35 The method of the present invention 50.4 1.89 0.56

2. Analysis according to amount of pretreatment solution

Once the air is passed through the membrane to remove the moisture of the membrane to an appropriate level, an appropriate amount of the pretreatment solution is added to the membrane, and the reaction is carried out for a certain period of time, thereby destroying the cells and the solid components, and freeing the nucleic acid from the cells.

The important condition here is the amount of pretreatment solution applied to the membrane.

Unlike existing nucleic acid extraction methods or commercial kits, the method proposed by the present invention does not include a separate procedure for concentrating nucleic acids. In addition, in the process of freeing the nucleic acid in the pretreatment solution, there is a possibility that the solid component remaining in the membrane in the urine component is melted and included in the pretreatment solution.

Therefore, when an excessive amount of the pretreatment solution is applied to the membrane, the concentration of the nucleic acid becomes too low, and the concentration required for the nucleic acid amplification reaction is not reached, so that the possibility that the amplification reaction does not occur increases. In addition, the amount of the inhibitor component that suppresses the nucleic acid amplification in the urine component increases, which may also interfere with the amplification reaction.

Conversely, if too little pretreatment solution is applied to the membrane, the yield of nucleic acid in the final product is lowered because the action of releasing the nucleic acid from the solid component remaining in the membrane is not sufficient. It is therefore important to apply an appropriate amount of pretreatment solution to the membrane.

The amount of the pretreatment solution suitable for the present invention was confirmed to be 100 to 200 占 퐇 in the 13 mm diameter membrane and 200 to 600 占 퐇 in the 25 mm diameter membrane.

In the case where the optimum condition was not proposed in the present invention, the concentration of the nucleic acid in the final solution was measured. As a result, the result could not be measured in the nano-drop apparatus and could not be used for the nucleic acid amplification experiment. If the amount of the pretreatment solution used is less than the amount of the pretreatment solution, the nucleic acid can not be sufficiently liberated. If a large amount of the nucleic acid is used, the amount of the impurity is increased and the nucleic acid concentration is lowered.

As shown in the results of FIG. 3 and FIG. 4, it can be seen that the pretreatment method of the optimized conditions in the present invention is a level suitable for use in nucleic acid amplification reactions such as PCR and LAMP as compared with other commonly used methods.

The primers used in the PCR reaction were human beta globin.

FW AGC CAC CAC TTT CTG AT BW GGG TTA AGG CAA TAG CAA T

The primer sequence information used in the LAMP reaction is as follows.

F3 AGC CAC CAC TTT CTG AT B3 GGG TTA AGG CAA TAG CAA T FIP ACC TCT TAT CTT CCT CCC ACA GGT GAA TTC TTT GCC AAA GTG BIP GCC TAG CTT GGA CTC AGA ATA ATC CTA ATA GCA GCT ACA ATC CAG LF CAA CGT GCT GGT CTG TG LB CAG CCT TAT CCC AAC CAT AA

The PCR conditions used for the amplification of the nucleic acid were as follows: initial denaturation at 95 ° C for 10 minutes, denaturation at 95 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds Was repeated 35 times and the final extension was carried out at 72 캜 for 5 minutes. LAMP experiments were carried out at 63 ° C for 30 min.

3, the results of lane 1 to lane 4 in the case of using the membrane treatment method developed in the present invention to permeate the urine membrane showed that a gene amplification product was detected in the pretreatment solution developed in the present invention, whereas Qiagen No product was found in the buffer product of the company. On the other hand, in both lane 5 and lane 6, where the pure nucleic acid was extracted from the urine, but not from the urine, both the pretreatment solution and the Qiagen product were used. The method developed by the present invention shows that the ability to extract nucleic acids purely from cells is low compared to Qiagen products, whereas the method of the present invention, which extracts urine samples simply using membranes, is effective in the pretreatment method proposed by the present invention .

4, when 2 μl of the lysate solution prepared using the pretreatment solution developed in the present invention was used as a template, the DNA band of the electrophoresis ladder form was clearly observed, whereas when using 8 μl of the lysozyme band, It did not appear. When lysate was made using Qiagen's product, no band appeared at 2 and 8 μl. When the LAMP reaction was performed after extracting the pure nucleic acid, DNA ladder was observed in the case of using Qiagen product, but no band appeared in the buffer developed in the present invention. It can be seen that the pretreatment solution developed in the present invention specifically acts on the method proposed in the present invention for treating urine using a membrane. It is also shown that the amount of lysate used in the LAMP reaction affects the LAMP reaction.

< Example  5> Analysis according to air injection amount

1. Air injection method used to remove residual urine in urine after passing urine

In the case of a hydrophilic membrane, urine remains in the membrane even after the urine has passed through the membrane. Therefore, in order to prepare the optimum conditions necessary for treating the cells and solid components remaining in the membrane by adding the pretreatment solution, Components must be able to remain constant.

If urine water remains in the membrane, the concentration of the pretreatment solution becomes low, which prevents the nucleic acid from separating from the cell through pretreatment. In order to suppress this, air pressure is applied to the membrane passing through the urine to remove the water present in the membrane, so the amount of air applied to the membrane and the time it takes to pass the air are important. If the amount and time of air are not adequate, the pressure on the membrane can not sufficiently remove the moisture contained in the pores of the membrane.

Conversely, if excess moisture is removed from the membrane, the solid component such as cells remaining in the membrane may become dry and the function of the pretreatment solution may not be exhibited. However, if the membrane is not extremely dry, it does not have a great effect.

The optimal air injection method of the present invention is to apply 10 to 20 cc of air to a 13 mm diameter membrane within 10 to 20 seconds. For 25 mm diameter membranes, 40 to 80 cc of air should be applied to the membrane within 10 to 20 seconds. If this calculated cross-sectional area in the film of 13mm diameter 0.007cc / mm ² / sec and a 0.008 cc / mm ² / sec in a 25mm diameter membrane.

2. Air injection method used to collect the pretreatment solution after pretreatment solution treatment

When the nucleic acid becomes sufficiently free, air pressure is applied again to separate the pretreatment solution present in the membrane and the nucleic acid dissolved in the membrane from the membrane and place in a clean separate container.

This solution can be used for nucleic acid amplification experiments or can be stored in a refrigerated or frozen state.

If the amount and time of the air are not appropriate at this stage, the pressure on the membrane can not sufficiently remove the pretreatment solution contained in the pores of the membrane. Therefore, in order to increase the amount of the final solution used for nucleic acid amplification, The air pressure of the condition should be applied.

The optimal air injection method of the present invention is to apply 10 to 20 cc of air to a 13 mm diameter membrane within 10 to 20 seconds. For 25 mm diameter membranes, 40 to 80 cc of air should be applied to the membrane within 10 to 20 seconds. If this calculated cross-sectional area in the film of 13mm diameter 0.007cc / mm ² / sec and a 0.008 cc / mm ² / sec in a 25mm diameter membrane.

< Example  6> Analysis of pretreatment solution by operating time

Another important factor in conjunction with the amount of pretreatment solution is the time of action of the pretreatment solution.

If the action time is too long, the possibility of nucleic acid destruction becomes high. The present invention does not include a component that inhibits DNase or RNase, which is an enzyme that destroys nucleic acids existing in the human body. Existing products or technologies contain DNase or RNase inhibitors to inhibit nucleic acid destruction. However, these components affect the amplification reaction of the nucleic acid, so the procedure for removing the nucleic acid must be performed.

In the present invention, these ingredients were excluded from the beginning in pursuit of cost reduction and simplicity of procedure. Therefore, if the pretreatment solution is used for a too long time, the nucleic acid is destroyed and the template to be used for the nucleic acid amplification reaction is highly likely to disappear.

On the other hand, if the reaction is performed for too short a time, the function of releasing the nucleic acid from the solid component containing the nucleic acid including the cell is insufficient, and the yield of the nucleic acid in the final product is lowered. This also results in lowering the nucleic acid concentration required for the nucleic acid amplification reaction so that the appropriate urine preprocessing process is not satisfied.

In the present invention, the appropriate time for the pretreatment solution action was confirmed to be 5 to 10 minutes.

In the case of the present invention, the concentration of the nucleic acid in the final product solution was measured. As a result, the concentration of the nucleic acid in the nano-drop apparatus could not be measured and the nucleic acid amplification experiment could not be used.

As a result, the urine pretreatment experiment method finally established in the present invention is as follows.

(1) When 1 to 30 ml of the collected urine sample is passed through a PDVF or NY membrane having a pore size of 0.2 to 10 μm and a diameter of 13 to 25 mm in a syringe, the liquid component of the urine is discharged through the membrane to the outlet do. Liquid components that pass through the membrane and exit the outlet are discarded.

(2) Solid components, including cells in the urine, can not pass through the membrane and remain on the membrane.

(3) In order to remove the liquid component of the urine remaining in the membrane, air is injected through the injection port by a syringe to push the liquid component present in the membrane, and the membrane is properly dried. The optimal air injection method of the present invention is to apply 10 to 20 cc of air to a 13 mm diameter membrane within 10 to 20 seconds. For 25 mm diameter membranes, 40 to 80 cc of air should be applied to the membrane within 10 to 20 seconds. If this calculated cross-sectional area in the film of 13mm diameter 0.007cc / mm ² / sec and a 0.008 cc / mm ² / sec in a 25mm diameter membrane.

(4) A pretreatment solution prepared by adding 40 to 60 mM NaCl, 40 to 60 mM Tris-HCl, 0.01 to 1% Triton X-100 and final pH of 6 to 9 to the membrane through an injection port and 100 to 200 And 200 to 600 μl for 25 mm diameter membranes.

(5) The pretreatment solution is allowed to react at room temperature for 5 to 10 minutes. The pretreatment solution destroys the solid components remaining on the surface of the membrane so that the nucleic acid is completely separated from the solid component such as cells and is dissolved in the pretreatment solution.

(6) When the reaction is completed, air pressure is again applied to the membrane using a syringe, and the pretreatment solution present in the membrane is completely pushed out through the outlet through the membrane. The optimal air injection method of the present invention is to apply 10 to 20 cc of air to a 13 mm diameter membrane within 10 to 20 seconds. For 25 mm diameter membranes, 40 to 80 cc of air should be applied to the membrane within 10 to 20 seconds. If this calculated cross-sectional area in the film of 13mm diameter 0.007cc / mm ² / sec and a 0.008 cc / mm ² / sec in a 25mm diameter membrane.

(7) The discharged pretreatment solution is a liquid component containing a nucleic acid, and this solution is collected in a clean container and can be directly applied to a nucleic acid amplification experiment without any special operation.

Claims (9)

(1) passing the separated urine through a hydrophilic membrane;
(2) applying air pressure to the membrane through which the urine of step (1) is passed;
(3) preparing a pretreatment solution containing 40 to 60 mM NaCl, 40 to 60 mM Tris-HCl, and 0.01 to 1% Triton X-100 and adjusted to a final pH of 6 to 9;
(4) injecting the prepared pretreatment solution into a film to which air pressure is applied in the step (2), and reacting; And
(5) A method for extracting nucleic acid from urine, comprising the step of applying air pressure to a membrane to which a pretreatment solution of step (4) has been reacted to obtain a nucleic acid extraction sample. The method according to claim 1, wherein the urine is 1 to 30 ml. The method of extracting nucleic acid from urine according to claim 1, wherein the hydrophilic membrane is polyvinylidene difluoride (PVDF) or nylon (NY) membrane. The method according to claim 3, wherein the hydrophilic membrane has a pore size of 0.2 to 10 μm and a diameter of 13 to 25 mm. The method according to claim 1, wherein the step of applying air pressure to the membrane is performed by injecting 10 to 80 cc of air for 10 to 20 seconds to apply an air pressure of 0.007 to 0.008 cc / mm ² / sec. Extraction method. delete The method of extracting nucleic acid from urine according to claim 1, wherein 100 to 600 μl of the pretreatment solution is injected in step (4). The method of extracting nucleic acid from urine according to claim 1, wherein the step (4) is carried out at room temperature for 5 to 10 minutes. The nucleic acid extraction method according to claim 1, wherein the nucleic acid is DNA.

Images