KR20150127917A

KR20150127917A - Method of extracting and amplifying nucleic acids by using magnetic bead

Info

Publication number: KR20150127917A
Application number: KR1020140054246A
Authority: KR
Inventors: 김태선; 송금수
Original assignee: (주)바이오메트릭스 테크놀로지
Priority date: 2014-05-07
Filing date: 2014-05-07
Publication date: 2015-11-18

Abstract

The present invention relates to a method for extracting and amplifying nucleic acids using magnetic particles, which repeats a DNA PCR process including the steps of denaturing DNA, coupling primers, and elongating DNA chains one or more times. The method of the present invention directly uses resultant denature DNA-magnetic particle attached materials without eluting denatured DNA/RNA from magnetic particles, and performs a step of coupling primers and a step of elongating DNA chains after washing the denature DNA-magnetic particle attached materials with a washing solution containing proteins. According to the present invention, first, DNA loss in samples can be prevented to perform PCR at high sensitivity and high efficiency for samples with low DNA content; second, the surface of solid support bodies can be non-specifically blocked with proteins to perform PCR at high sensitivity and high efficiency even under the presence of solid support bodies; and third, DNA denaturing process and PCR process can be performed within a single identical container to prevent DNA from being polluted.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for extracting and amplifying a nucleic acid using magnetic particles,

The present invention relates to a nucleic acid-based measurement technique with high sensitivity and efficiency, and more particularly to a method for detecting DNA-magnetic particles attached to a denatured DNA-magnetic particle deposit resulting from the denaturation step, The present invention relates to a nucleic acid extraction and amplification method comprising washing a denatured DNA-magnetic particle attachment with a protein-containing wash solution followed by a primer binding step and a DNA chain extension step, and a method for diagnosing virus infection using the same.

Research in biotechnology using DNA / RNA has been steadily developed since the early 1900s for DNA / RNA sequencing, as well as for the determination of specimens, the presence or absence of disease, and the characterization of DNA / RNA. In recent years, And a method for detecting it.

Molecular diagnostic tests, including genetic diagnostic tests and immunohistological tests, are used to evaluate genes, metabolic functions, drug metabolism, and disease-inducing relationships based on biomarkers such as nucleic acids (DNA, RNA) Currently, it is used in the same sense as nucleic acid diagnostics, which can analyze and detect nucleic acid, which is a genetic information material derived from a very small amount of infectious agent, and diagnose infection.

Nucleic acid diagnosis is based on PCR, which is based on the detection, amplification, and analysis of nucleic acid, i) virus quantification and gene analysis, ii) sexually transmitted diseases and infection tests, iii) diagnosis, monitoring and prognosis, ) And genetic tests used in drug genetics and drug genomics.

The core of the technology using DNA / RNA is the nucleic acid extraction process, which extracts and purifies the nucleic acid from the sample or the sample. Efforts and studies have been continuing since the early 1900s. The nucleic acids extracted from the specimens were removed from a large amount of salt and protein impurities that impeded the progress of the subsequent process, and concentrated in a small amount of buffer, thereby enabling more accurate and effective analysis experiments. The introduction of the PCR method, a gene amplification technique developed in 1983, enables rapid and effective analysis of the purified nucleic acid due to its high sensitivity and specificity, so that the nucleic acid-based analysis technique is very resilient .

Many nucleic acid extraction methods have been developed including phenol-chloroform precipitation, Boom technology (solid based nucleic acid purification), SPRI (solid phase reversible immobilization) and CST (charge switch technology) Boom technology to immobilize nucleic acid on silica surface using chaotropic salt is mainly used, and it is mainly used as a main technology of DNA / RNA isolaiton kit sold by Qiagen and MN. The chaotropic salt is a typical water molecule network breaker that induces direct adsorption of DNA on the surface of a solid substrate to increase the recovery rate of the nucleic acid, and at the same time, the cell membrane (cell debris) membrane and other proteins can be dissolved to obtain high-purity nucleic acids.

However, chaotropic salts are highly toxic and cause degradation of DNA polymerase when they remain in the DNA / RNA extraction solution, thus acting as a powerful PCR inhibitor. Therefore, when a chaotropic salt is used, a washing process is indispensable, so two or three washing steps are necessary. This method is mainly refined using columns or magnetic particles, and DNA / RNA is removed from the column or magnetic particles on the aqueous solution due to refinement of the nucleic acid in an open plastic ware, high surface area, The elution or elution process is necessarily required.

The volume of the sample containing the eluted DNA / RNA is in the range of 100-200 μl, and the sensitivity is lowered because only a small amount of 2 ~ 10 μl is consumed for the PCR analysis. The amount of DNA / RNA is very small The efficiency of PCR decreases. In addition, in the elution process, loss of whole DNA / RNA due to the desorption efficiency of adsorbed DNA / RNA can not be avoided due to a partial loss of DNA / RNA.

In addition, when nucleic acid amplification processes such as PCR are performed using the extracted nucleic acid, particularly when a plurality of samples are used, frequent migration of the sample frequently results in false-positive results due to cross-contamination.

In order to overcome these problems, an automatic device for preventing an operator's handling error has been proposed. However, the automated devices developed so far are mainly limited to the nucleic acid extraction function, and even if the nucleic acid extraction process and the nucleic acid amplification process are integrated, the movement of the nucleic acid extraction solution and the complex mechanical movement are required, However, the migration of the sample, which requires transferring the eluted nucleic acid to the PCR tube, and the cross-contamination caused by the migration of the sample still remain as a serious problem.

In the conventional PCR method, there is a need for an improved PCR method for solving the problems of low sensitivity and low efficiency caused by using only a part of the DNA / RNA present in the sample and the problem of contamination due to sample migration.

In a PCR method for extracting and amplifying nucleic acid using a solid support such as magnetic particles, the problem of sensitivity and low efficiency caused by using only a part of DNA / RNA present in the sample and the problem of contamination due to sample migration are solved To develop an improved PCR method.

The present inventors have found that, in a nucleic acid extraction and amplification method including a PCR process including a DNA denaturation step, a primer binding step and a DNA chain lengthening step using magnetic particles as a solid support,

(1) denaturing the DNA in the presence of magnetic particles to attach the denatured DNA / RNA to the magnetic particles,

(2) When washing the denatured DNA / RNA-adhered magnetic particles (hereinafter also referred to as "adherent of denatured DNA / RNA and magnetic particles" or simply "denatured DNA-magnetic particle adhere"), Lt; RTI ID = 0.0 > of protein, < / RTI >

(3) By performing the primer binding step and the DNA chain extension step using the resulting denatured DNA-magnetic particle attachment itself without eluting the denatured DNA / RNA from the magnetic particles after the washing,

First, since loss of DNA in a sample can be prevented, PCR can be performed with high sensitivity and high efficiency even for a sample having a low DNA content,

Second, by non-specifically blocking the surface of the solid support with a protein, DNA PCR can be performed with high sensitivity and efficiency even in the presence of a solid support,

Third, we found that DNA contamination can be prevented because the DNA denaturation step and the PCR step can be performed in the same container, and the present invention has been completed.

According to the present invention, it is possible to perform DNA PCR with high sensitivity and efficiency even for a sample having a low DNA content, and to perform a DNA PCR process with high sensitivity and efficiency even in the presence of a solid support, Denaturation and PCR can be carried out in a single container, thereby preventing DNA contamination.

Figure 1 schematically illustrates the integration of nucleic acid extraction and amplification processes in accordance with embodiments of the present invention;
FIG. 2 is a view showing the results of PCR reaction involving magnetic particles using BMT 9G DNA KIT due to integration of nucleic acid extraction and amplification according to an embodiment of the present invention; FIG.
FIG. 3 is a view showing a result of PCR according to an amount of magnetic particles used according to an embodiment of the present invention, using BMT 9G DNA KIT; FIG.
Fig. 4 is a view showing the results of PCR obtained by varying the concentration of the added skim milk using BMT 9G DNA KIT and showing the electrophoretic pattern; Fig.
5 is a view showing the result of PCR obtained by fixing the concentration of skim milk to 0.05% and changing the number of magnetic particles inserted in the PCR reaction using BMT 9G DNA KIT;
FIG. 6 shows the results of PCR using BMT 9G DNA KIT when the amount of magnetic particles inserted into the PCR reaction was changed to 1 mg / 20 μl and 2.5 mg / 20 μl when 0.05% and 0.1% of skim milk were added. Fig.
FIG. 7 is a graph showing the results of PCR obtained for several samples by using a BMT 9G DNA KIT and fixing the concentration of the skim milk to 0.1% and the amount of the magnetic particles to 2.5 mg / 20 μl.
In each figure, that is, each color has a fluorescence intensity, and the specific fluorescence intensity has the order and intensity of white>red>yellow> blue = 65000>50000>40000> 30000 >> 10000. Even in the same green, bright green has a strength of about 35000 and dark green has a strength of about 20000, so bright colors show higher intensity.

A first object of the present invention is to provide a nucleic acid extracting and amplifying method comprising at least one PCR of a DNA comprising a DNA denaturation step, a primer binding step and a DNA chain lengthening step, There is provided a nucleic acid extraction and amplification method comprising:

(1) denaturing the DNA in the presence of a solid support such as magnetic particles, attaching denatured DNA / RNA to the surface of the magnetic particles,

(2) washing the denatured DNA-magnetic particle attachment resulting from step (1) with a protein-containing washing solution,

(3) performing the primer binding step and the DNA chain extension step using the resulting denatured DNA-magnetic particle attachment in step (2) as such.

According to one embodiment of the present invention, step (2) may be performed in detail as follows:

(2-1) Denaturing DNA-The protein-containing washing solution is added to a reaction vessel containing magnetic particle attachment,

(2-2) The reaction vessel described above is stirred or shaken to wash the denatured DNA-magnetic particle attachment described above with the above-mentioned protein-containing washing solution,

(2-3) The magnetic particles in the mixture obtained in (2-2) above are fixed with magnets,

(2-4) The washing solution is separated and removed from the mixture obtained in (2-3) above,

(2-5) In some cases, the above steps, for example, steps (2-1) to (2-4) are repeated one or more times.

According to one embodiment of the present invention, the above-mentioned solid support, specifically, the magnetic particle may have at least one functional group which can be selected from the group consisting of a siloxane group, an amino group and a hydroxyl group on the surface, Amines, polystyrenes, polypropylenes, and the like. The size (diameter) of the above magnetic particles may be 0.1 to 30 탆, specifically 0.2 to 25 탆, and preferably 0.4 to 20 탆. The amount or content of the magnetic particles may be selected in the range of 0.10 to 10 mg / 20 μl, specifically 0.25 to 5 mg / 20 μl, preferably 0.5 to 2.5 mg / 20 μl. The amount or content of the magnetic particles is selected to efficiently perform the amplification process of the DNA including the PCR process. In some cases, the magnetic particles may be used in an amount of 2.5 to 30 mg / It is also within the scope of the present invention to use a high content of 5 to 20 mg / 20 μl.

According to one embodiment of the present invention, in the protein-containing wash solution described above, the protein may be selected from the group consisting of BSA, skim milk and casein, and its content (concentration) , But may have an h concentration of 0.01 to 0.1% (w / v), specifically 0.02 to 0.1% (w / v), preferably 0.03 to 0.08% (w / v) .

According to one embodiment of the present invention, the DNA chain lengthening step can be carried out using a DNA amplification solution selected from the group consisting of PCR, RT-PCR, TMA, NASAB, SDA and RCA solutions.

According to one embodiment of the present invention, the result obtained by the nucleic acid extraction and amplification reaction is subjected to gel electrophoresis, enzyme linked gel assay (ELGA), electrochemiluminescent (ECL) or BMT 9G DNA KIT and BMT Genotyping 9G Membrane KIT. &Lt; Desc / Clms Page number 7 >

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. It is to be understood that the following description is of a preferred embodiment of the present invention and that the present invention is not limited thereto.

In the context of the present invention, the nucleic acid may refer to DNA, RNA, fragments or oligomers thereof, and the method of the present invention may be applied to all of them, and thus the "DNA chain extension step" , "DNA-magnetic particle attachment (binding body)" and the like can be used by replacing DNA with RNA. Further, when the method of the present invention is applied to RNA, a step of transforming double-stranded DNA into a single strand, for example, DNA denaturation, may be omitted.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic illustration of the integration of nucleic acid extraction and amplification processes in accordance with embodiments of the present invention.

According to the drawing, the sample, the nucleic acid extracting solution and the washing solution are added by a nozzle in one chamber containing magnetic particles, or the solution is removed so that the magnetic particles adsorbing the nucleic acid do not move out of the chamber. Also, a method of increasing the sensitivity and accuracy by using the whole nucleic acid obtained from the sample in PCR is omitted, since the PCR solution is added to the nucleic acid-absorbed magnetic particles through the nozzle and the elution process is omitted.

A nucleic acid-containing sample is added to a chamber to which nucleic acid extraction solution and magnetic particles are added, followed by nucleic acid extraction. Nucleic acids isolated from bacteria, viruses and animal cells by nucleic acid extraction solution are adsorbed on magnetic particles. The magnetic particles adsorbed by the nucleic acid by the magnetic bar located outside the chamber are fixed in the chamber and all of the solution excluding the magnetic particles is removed by suction. The washing solution is injected into the chamber through the nozzle to purify the nucleic acid adsorbed to the magnetic particles, and a predetermined protein is added to the washing solution. After the washing process, the solution is removed in the same manner as in the method of removing the extraction solution, and only the magnetic particles adsorbed on the purified nucleic acid remain in the chamber. Thereafter, the PCR solution is added to the chamber, and the PCR reaction is performed by the heater located at the bottom of the chamber.

The above-mentioned invention suggests a processing method that can reduce contamination, improve sensitivity and accuracy by integrating a series of processes of nucleic acid extraction and amplification.

1. Integration process of nucleic acid extraction and amplification

In the present invention, the principle of the integrated process of nucleic acid extraction and amplification can be described as follows:

Currently, the nucleic acid extraction kit uses magnetic particles as the column or solid support for adsorption, separation and purification of nucleic acid. The use of columns and magnetic particles has the advantage of easy extraction and purification because nucleic acids can be adsorbed on a solid substrate to allow nucleic acid separation from a large number of samples. However, such a solid-based nucleic acid extraction method is required to be eluted again in a liquid phase because it is necessary to convert it into a liquid phase in order to analyze and measure using a subsequent process, for example, PCR.

When the adsorbed nucleic acid is desorbed again, the elution process is losing purified nucleic acid according to desorption efficiency. In addition, most of the nucleic acids extracted from the sample are wasted because a minimum amount of the solution required for elution is required to be at least about 100 μl, but a subsequent process, for example, PCR, uses only a maximum of 10 μl at a minimum of 2 μl.

Accordingly, the present invention provides an integrated process capable of performing simple and rapid measurement by adding a PCR solution to a magnetic particle by omitting the elution step after completion of lysis, nucleic acid adsorption and washing in the nucleic acid extraction process using magnetic particles Respectively.

The whole amount of the nucleic acid adsorbed on the magnetic particles obtained from the sample is immediately added to the PCR solution to perform the PCR reaction. At this time, the nucleic acid is desorbed into the aqueous phase effectively because it proceeds at a high temperature of 94 ° C. in the first denaturation step, and this is used for the PCR reaction immediately. This has high sensitivity and accuracy because it uses the entire amount of nucleic acid in the sample and does not lead to false positive results due to contamination due to sample migration since the nucleic acid-adsorbed magnetic particles do not migrate in one chamber.

2. Optimization process for nucleic acid amplification

The present invention proposes a method of adding a predetermined protein in the final washing step of the nucleic acid extraction process to solve the problem of inhibiting PCR reaction on a large amount of magnetic particles. The protein added to the final washing step nonspecifically adsorbs to the surface of the magnetic particles other than the portion where the nucleic acid is adsorbed, thereby blocking the elements inhibited by the PCR reaction, thereby leading to more efficient PCR reaction.

The above proteins may be selected from the group consisting of BSA (bovine serum protein), skim milk and casein, and may be selected from the group consisting of 0.005 to 0.2% (w / v), specifically 0.01 to 0.1% (w / v), preferably 0.02 to 0.08% (w / v). Alternatively, the use of gelatin or a commercially available non-animal protein blocking agent as the aforementioned protein may be included in the scope of the present invention.

Proteins such as BSA, skim milk and casein described above are proteins used as a blocking buffer or a blocking agent in Western blotting for analyzing the presence or amount of a specific protein, the membrane is coated with a protein on the surface of the unblotted membrane to block the 1st Ab or 2nd Ab to prevent nonspecific binding. However, such blocking agents are not used in Southern blotting to detect DNA, and thus the use of such blocking agents and their effects in PCR using solid supports has not been known until now. However, when PCR is carried out using a solid support (eg micro fluidic chip), the surface of the chip solid may be coated with BSA or BSA may be added to the PCR solution. However, as a blocking agent as in the present invention, There is a difference.

When the DNA amplification reaction such as the primer binding step and the DNA chain lengthening step is carried out in the presence of the solid support, the present inventors have found that when the DNA amplification reaction is disturbed or inhibited by the solid support, if the surface of the solid support is blocked with the above- It was confirmed that disturbance or inhibition of the DNA amplification reaction by the solid support can be suppressed.

On the other hand, according to one variant of the present invention, the method of the present invention can be carried out in such a manner that the solid support is separated from the washing solution by centrifugation and the washing solution is removed by decanting or pipetting.

According to a further variation of the present invention, when the solid support can be separated from the washing solution by centrifugation, the method of the present invention can be carried out without the magnetic particles as described above. In this case, the step (2) may be performed in detail as follows:

(2-3) The mixture resulting from (2-2) was centrifuged,

(2-4) The washing solution is separated and removed from the mixture obtained in the above (2-3) by slanting and / or pipetting,

(2-5) In some cases, the above steps (2-1) to (2-4) are repeated.

PCR methods using magnetic particles can be found in many literatures, and those not specifically described in the present invention can use those described in the above-mentioned documents.

According to the method of the present invention, not only the solid support to which the denatured DNA is attached is washed with a solution containing the specific protein, but also the primer binding step and the DNA chain extension step by using the denatured DNA as it is, By performing the same subsequent PCR process, the following advantages can be obtained:

First, since there is no elution step for separating the denatured DNA / RNA from the solid support, the loss of DNA can be prevented. Therefore, the PCR can be performed with high sensitivity and high efficiency even for a sample having low DNA content,

Second, by blocking the surface of the solid support with non-specific binding to the protein using the protein-containing washing solution, the PCR process of the DNA can be performed with high sensitivity and efficiency even in the presence of the solid support,

Third, the denatured DNA / RNA and attachment of the solid support described above can remain in the reaction vessel after the reaction and washing step, and the subsequent PCR reaction can proceed without movement or alteration of the reaction vessel. As a result, Denatured and subjected to a PCR reaction, so that contamination of the DNA can be prevented.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the present invention is not limited thereto.

Reference example One

HPV (human papilloma virus) was used as a nucleic acid-containing sample, and Nucleic Acid Extraction (Magnetic bead) kit [He Nan Hui Nano Science And Technology Co., LTD. China] was used.

According to the manual, the PCR procedure consists of (i) DNA denaturation, (ii) elution of denatured DNA, (iii) primer annealing, and (iv) chain extension.

The above-mentioned elution includes a washing process of the DNA denaturation step unreacted material, a heating process for mixing and separation of the separation material. The denatured DNA is separated from the magnetic particles in the elution step and then amplified through the primer binding step and the DNA extension step.

(1) Control group

As a control, the PCR procedure was carried out four times in accordance with the nucleic acid extraction procedure specified in the manual of the above-mentioned kit.

Since the amplified nucleic acid is used by mixing a hybridization solution with the PCR solution, there is no need to separate and purify the nucleic acid separately, BMT HPV 9G DNA KIT [Biometrics Technology; Korea] were used to compare fluorescence intensities. The fluorescence intensities are in the order of white-red-yellow-green-blue in strong order.

Photographs 1 to 4 on the left side (without bead) of FIG. 2 show fluorescence images measured on the amplified DNA.

(2) Experimental group

As an experimental group, except for carrying out the washing step in the elution step and then omitting the mixing step and the heating step, the amplified step was carried out without separating the denatured DNA from the magnetic particles. PCR was carried out four times using the detailed extraction method as it was.

The extracted DNA was denatured in the presence of magnetic particles, and the resultant denatured DNA-magnetic particle adherent was washed, and a small amount of Tris-HCl (pH 8.0) buffer was added thereto to float. The suspended magnetic particles were quantitated and analyzed using a PCR premix tube (Bioneer; Korea) and PCR was performed (once) to amplify the nucleic acid.

The amplified nucleic acid Without separate isolation or purification, BMT HPV 9G DNA KIT [Biometrics Technology; Korea] were used to compare fluorescence intensities. The fluorescence intensities are in the order of white-red-yellow-green-blue in strong order.

Photographs 1 to 4 of the right side of FIG. 2 (with bead) show fluorescence images measured on the amplified DNA.

Compare results

Fig. 2 shows the fluorescence photographs (with bead) (experimental group) obtained from the PCR result in the state of denatured DNA-magnetic particle attachments and the fluorescence photographs (without bead) obtained in the PCR result in the state of the denatured DNA only without the magnetic particles ).

For each of the four samples, the blue spot of the control group was not expressed in the test group in the first sample, while the fluorescence difference was the least decreased in the control group white to the red of the test group in the third sample, but in the second and fourth samples In the control group, fluorescence decreased rapidly in the green color of the test group.

As a result, the sensitivity generally decreases in PCR (experimental group) performed in the presence of magnetic particles.

Reference example 2a and 2b

Except that the amount of the magnetic particles was changed to 0 mg (control group), 0.25 mg / 20 μl, 0.5 mg / 20 μl, 1 mg / 20 μl, 1.5 mg / 20 μl and 2.5 mg / 20 μl, respectively PCR was carried out in the same manner as in Experimental group 1. The control group (0 mg dose) means that the PCR process was performed in the same manner as the control group of Reference Example 1.

FIG. 3 is a graph showing the PCR results of Reference Example 2a (Sample 1) and Reference Example 2b (Sample 2). When the amount of magnetic particles used was 0.25 mg / 20 μl, the result was slightly lower than that of the control, . However, when the used amount is more than 1 mg / 20 μl, the PCR efficiency is drastically reduced as compared with the control group, and when the use amount is 2.5 mg / 20 μl, the results are not obtained properly. Therefore, it can be seen that the presence of the magnetic particles does not act as an impediment factor of the efficiency of the PCR when the magnetic particles are used at a rate of 0.25 mg / 20 μl or less or below.

Example One

Using a washing solution containing magnetic particles in an amount of 0.5 mg and containing a specific protein (eg skim milk) in the elution step at a concentration of 0%, 0.01%, 0.03%, 0.05% or 0.1% (w / v) PCR was carried out in the same manner as in Reference Example 1. < tb > < TABLE > As a control, PCR was carried out in the absence of magnetic particles and the result obtained using a washing solution containing no skim milk (same as the control in Reference Example 2a) was used.

4A shows that the fluorescence intensity of the control group is white, but when the protein is not used, the sensitivity is greatly reduced to green. However, when the washing solution containing the skim milk protein in the amounts of 0.01%, 0.03%, 0.05% and 0.1% , It is possible to obtain similar or similar sensitivities to the control group.

FIG. 4B shows the PCR result of FIG. 4A confirmed by electrophoresis. As a result, when the 0.05% skim milk protein was used, the most distinct PCR band was confirmed.

Example 2

(Eg, skim milk) was used in the elution step by using magnetic particles in an amount of 0.5 mg / 20 μl, 1 mg / 20 μl, 1.5 mg / 20 μl, 2.5 mg / 20 μl and 5 mg / PCR was carried out in the same manner as in Example 1, except that a washing solution containing 0.05% (w / v) was used. As a control, PCR was carried out in the absence of magnetic particles and the results obtained using a washing solution containing no skim milk were used.

FIG. 5 shows that when using the washing solution containing 0.05% of the skim milk, the use of the magnetic particles in an amount of 1 mg / 20 μl resulted in the same or similar result as the control. These results indicate that using a washing solution containing skim milk can use magnetic particles in an amount of up to 1 mg / 20 가 instead of 0.25 mg / 20 ㎕ or less, thereby increasing nucleic acid extraction efficiency The nucleic acid amplification efficiency can be increased, and as a result, the measurement sensitivity can be improved.

Studies of the prior art have shown that when performing PCR in the presence of magnetic particles, the PCR efficiency is impaired due to the large surface area of the magnetic particles (see Example 1). Although the presence of a small amount of magnetic particles in the PCR process is acceptable, the sensitivity of the PCR is low because the amount of the magnetic particles is small, and the presence of a large amount of magnetic particles causes a problem of deteriorating the sensitivity as well as the sensitivity of the PCR. Therefore, it was essential to carry out PCR after separating and eluting the denatured DNA from the magnetic particles.

According to the present invention, proteins such as BSA, skim milk and casein can be added to the washing process to nonspecifically bind the proteins to the magnetic particles, thereby blocking the inhibitory effect on the subsequent PCR efficiency.

Example 3

The magnetic particles were used in the amounts of 1 mg / 20 μl and 2.5 mg / 20 μl, respectively, and a washing solution containing specific proteins (eg skim milk) at concentrations of 0.05% and 1% (w / v) PCR was carried out in the same manner as in Example 1. As a control, PCR was carried out in the absence of magnetic particles, and the results obtained using a washing solution containing no skim milk (the same as the control in Example 2) were used.

FIG. 6 shows that, when a small amount (1 mg / 20 μl) of magnetic particles is used, a better result can be obtained by using a cleaning solution containing a low concentration (0.05%) of skim milk, (0.1%) of skim milk was used when a large amount (2.5 mg / 20 μl) was used as a washing solution.

Thus, it is shown that to increase the amount of magnetic particles to obtain high measurement efficiency, a washing solution containing more protein should be used. When a small amount of magnetic particles are used, there is a high possibility that the remaining proteins are adsorbed nonspecifically on the surface of the magnetic particles, and such unbound residue proteins are released into the PCR solution and can act as an inhibitor to the PCR. However, when a large amount of magnetic particles are used, they are nonspecifically adsorbed on the surface of magnetic particles and the amount of remaining proteins is very small, so that they are unlikely to be released into the PCR solution. Therefore, a higher concentration of protein is required and can be used to perform the integrated process of nucleic acid extraction and amplification using a large amount of magnetic particles.

Example 4

For four nucleic acid-containing samples, use a washing solution containing magnetic particles in an amount of 2.5 mg / 20 μl and a specific protein (eg skim milk) at a concentration of 1% (w / v) in the elution step PCR was carried out in the same manner as in Example 1. As a control, for each nucleic acid-containing sample, PCR was performed in the absence of magnetic particles and the results obtained using a wash solution containing no skim milk were used.

FIG. 7 shows that Sample 1 has increased from blue to green, Sample 2 has increased from green to white, and Sample 3 and Sample 4 have not changed their results to blue and white, respectively.

The results of the present invention show that the PCR is carried out in the presence of magnetic particles according to the present invention and the sensitivity is much improved when the protein-containing washing solution is used, rather than performing the conventional general nucleic acid extraction and amplification process. It is also possible to increase the convenience and speed of the process by reducing the number of processes and to eliminate nucleic acid extraction / amplification that can improve the sensitivity and accuracy by using the entire amount of the nucleic acid obtained from the sample without deriving the false- The development of the integrated process of

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

A nucleic acid extracting and amplifying method comprising repeating a PCR of a DNA comprising a DNA denaturation step, a primer binding step and a DNA chain lengthening step one or more times, comprising the steps of:
(1) denaturing the DNA in the presence of a solid support such as magnetic particles, attaching the denatured DNA to the surface of the magnetic particles,
(2) washing the denatured DNA-magnetic particle attachment resulting from step (1) with a protein-containing washing solution,
(3) performing the primer binding step and the DNA chain extension step using the resulting denatured DNA-magnetic particle attachment in step (2) as such.

The nucleic acid extracting and amplifying method according to claim 1, wherein the solid support has at least one functional group selected from the group consisting of a siloxane group, an amino group and a hydroxyl group on the surface.

The method of claim 1, wherein the solid support is coated with at least one material selected from the group consisting of silica, carbohydrate, amine, polystyrene, and polypropylene.

The nucleic acid extracting and amplifying method according to claim 1, wherein the magnetic particles are used in an amount of 0.25 to 5 mg / 20 μl.

The method of extracting and amplifying nucleic acid according to claim 1, wherein the magnetic particles are used in an amount of 5 to 20 mg / 20 μl.

The method according to claim 1, wherein the protein in said protein-containing wash solution is selected from the group consisting of BSA, skim milk, and casein.

The method according to claim 5, wherein the protein is contained in an amount of 0.01 to 0.1% (w / v).

The method according to claim 1, wherein the DNA chain extension step is performed using a DNA amplification solution selected from the group consisting of PCR, RT-PCR, TMA, NASAB, SDA and RCA solutions.

2. The method of claim 1, wherein the nucleic acid extraction and amplification reactions are performed using gel electrophoresis, enzyme-linked gel assay (ELGA), electrochemiluminescent (ECL) or BMT 9G DNA KIT and BMT genotyping 9G Membrane KIT Wherein the nucleic acid is detected by a method selected from the group consisting of:

The method according to claim 1, wherein the step (2) comprises the steps of:
(2-1) adding a protein-containing washing solution to a reaction vessel containing the denatured DNA-magnetic particle attachment resulting from the step (1)
(2-2) The reaction vessel described above is stirred or shaken to wash the denatured DNA-magnetic particle attachment described above with the above-mentioned protein-containing washing solution,
(2-3) The magnetic particles in the mixture obtained in (2-2) above are fixed with magnets,
(2-4) Separation and removal of the washing solution from the mixture obtained in (2-2) above.

The nucleic acid extracting and amplifying method according to claim 1, wherein the nucleic acid is RNA.

A method for diagnosing a viral infection using the nucleic acid extraction and amplification method of claim 1.