JP2011097828A - Method for pretreatment of whole blood sample and nucleic acid amplification method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply coagulating contaminants without the need of any centrifugal separation treatment, which can be employed as a pretreatment for amplifying a nucleic acid in a whole blood sample and a nucleic acid amplification method which can amplify a nucleic acid in a whole blood sample conveniently with high efficiency. <P>SOLUTION: The method for pretreatment of nucleic acid in a whole blood sample includes (a) a step of diluting the whole blood sample with a diluent solvent containing a polyvalent cation and (b) a step of heat-treating the whole blood sample diluted in the step (a). The nucleic acid amplification method includes carrying out a nucleic acid amplification reaction by using a treatment solution pretreated by the pretreatment method for a whole blood sample. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention provides a method for simply performing a pretreatment for amplifying a nucleic acid of a whole blood sample without performing a centrifugation treatment, and a nucleic acid amplification using a whole blood sample pretreated by the method. On how to do.

With recent advances in genetic engineering technology and molecular biology, analysis of nucleic acids contained in samples has been widely performed not only in the field of academic research but also in the medical field. For example, in order to diagnose genetic diseases, cancer, infectious diseases, lifestyle-related diseases, etc., nucleic acids such as genomic DNA and mRNA in biological samples are analyzed. Usually, since the amount of nucleic acid contained in a biological sample is very small, analysis is often performed by amplifying a target nucleic acid to be analyzed.
The biological sample contains various substances including proteins, but these substances can be an inhibitory factor for nucleic acid amplification reaction. In particular, many proteins have nucleolytic activity and inhibitory activity such as enzymes used for nucleic acid amplification. In order to amplify a target nucleic acid with high accuracy and high sensitivity, inactivation of proteins in biological samples or Removal is preferably performed. For this reason, nucleic acid amplification is usually performed using a biological sample that has been subjected to pretreatment such as nucleic acid extraction.

  Various methods have been disclosed for such pretreatment methods. For example, there is an extraction method using a silica gel particle column. Here, the extraction method using a silica gel particle column refers to (a) adding a surfactant to a biological sample to lyse cells and digesting protein with proteinase K. (b) biological sample after the digestion treatment Is passed through a silica gel particle column to adsorb the nucleic acid to the silica gel particle, and (c) the nucleic acid is then extracted by eluting the nucleic acid from the column using an eluent. This method using a silica gel particle column is widely used by commercially available kits and the like, and a fully automatic machine for nucleic acid extraction using silica gel particles as magnetic particles is also commercially available. A commercially available kit for purification using this silica gel particle column is simple and can obtain a nucleic acid having a very high purity, and is therefore widely used in genetic testing using blood. However, there is a problem that the silica gel particle column is expensive and very expensive.

  Moreover, as a method for removing contaminants other than nucleic acids, in particular, substances that inhibit enzyme reactions such as nucleic acid amplification reactions, from a biological sample containing nucleic acids, for example, (1) removing at least one contaminant from a nucleic acid-containing sample A method for removing, comprising: (a) contacting the nucleic acid-containing sample with at least one flocculant to form a flocculant precipitate; and (b) separating the nucleic acid from the flocculant precipitate. The method including the process of performing is disclosed (for example, refer patent document 1). The method may include purifying or isolating the nucleic acid after step (b) and including degrading, denaturing or destroying the nucleic acid-containing sample prior to contact with the aggregating agent. May be. Examples of the flocculant include a cationic chemical substance, an anionic chemical substance, an amphoteric chemical substance, an uncharged chemical substance, or a combination thereof. In addition, (2) As a method for extracting bacterial DNA contained in soil from soil, pretreatment with aluminum ions or trivalent iron ions is performed, and nucleic acid extraction is performed to remove soil-derived inhibitors. (For example, refer nonpatent literature 1).

On the other hand, as a cheaper method, there is a boiling method in which a biological sample is simply boiled, and contaminants such as proteins in the biological sample are coagulated and separated from nucleic acids (see, for example, Non-Patent Document 1). . As a method applying this boiling method, for example, (3) a pretreatment method for amplifying a nucleic acid in a whole blood sample, (i) a step of diluting the whole blood sample, and (ii) diluted There is a pretreatment method including a step of heat treating a whole blood sample and (iii) a step of centrifuging the sample after the heat treatment (see, for example, Patent Document 2). For biological samples with a lot of contaminants, such as blood, where the nucleic acid content is very small, the accuracy of the nucleic acid obtained is not sufficient simply by boiling, as in the boiling method, and accurate testing is difficult However, as in the method of (3) above, the substance that inhibits the nucleic acid amplification reaction can be efficiently removed by centrifuging the contaminants after coagulation, so that a more accurate test can be performed. It becomes possible to do.
Special table 2008-500066 gazette JP 2006-187221 A Braid, 2 others, Journal of Microbiological Methods, 2003, Vol. 52, p389-393 Protein Nucleic Acid Enzyme, Kyoritsu Shuppan, 1996, Vol. 41, No. 5, p453-456

  In the case of processing a large number of specimens such as clinical examinations, it is preferable from the viewpoint of work efficiency that the number of examination steps is small and no special apparatus is required. Moreover, if the operation of each process is simple, it is easier to automate the inspection, which is more preferable. However, the methods (1) and (2) have a problem that the number of steps is large, the work is complicated, and the work efficiency is not sufficient. Further, although the method (3) has a smaller number of steps than the methods (1) and (2), there is a problem that the operation is complicated because the method includes a centrifugal separation process. Further, when the process including the centrifugation operation is automated, there is a problem that the configuration of the apparatus becomes complicated and the apparatus cost increases.

  The present invention is a pretreatment for amplifying a nucleic acid of a whole blood sample, a method capable of easily coagulating contaminants without performing a centrifugation treatment, and a nucleic acid in a whole blood sample simply and efficiently. An object of the present invention is to provide a nucleic acid amplification method that can be amplified well.

  As a result of diligent research to solve the above-mentioned problems, the present inventor easily simplifies contaminants such as proteins by diluting a whole blood sample with a diluting solvent containing polyvalent cations such as magnesium ions. The present invention was completed by finding that it can be inactivated and that the obtained treatment solution can be subjected to a nucleic acid amplification reaction without being subjected to a centrifugal separation treatment.

That is, the present invention
(1) A pretreatment method for amplifying a nucleic acid in a whole blood sample, comprising: (a) diluting the whole blood sample with a diluting solvent containing a polyvalent cation; and (b) step ( a step of heat-treating the whole blood sample diluted in a), and a pretreatment method of the whole blood sample,
(2) The pretreatment method of a whole blood sample according to (1), wherein the polyvalent cation is an alkaline earth metal ion or an aluminum ion,
(3) The pretreatment method of a whole blood sample according to (1), wherein the polyvalent cation is an aluminum ion or an iron ion,
(4) The pretreatment method of a whole blood sample according to any one of (1) to (3), wherein the temperature of the heat treatment in the step (b) is 50 to 100 ° C.
(5) The pretreatment method of a whole blood sample according to (1) or (2), wherein the temperature of the heat treatment in the step (b) is 50 to 90 ° C.,
(6) A nucleic acid amplification method comprising performing a nucleic acid amplification reaction using a treatment solution pretreated using the whole blood sample pretreatment method according to any one of (1) to (5) above,
The purpose is to provide.

  By the pretreatment method of the whole blood sample of the present invention, impurities in the whole blood sample can be inactivated easily without performing a centrifugation treatment. For this reason, nucleic acid amplification can be performed with high accuracy and efficiency by using the treatment liquid obtained by the whole blood sample pretreatment method of the present invention.

  The whole blood sample subjected to the whole blood sample pretreatment method of the present invention may be whole blood collected from an animal. For example, it may be a whole blood sample derived from a human, a whole blood sample derived from a laboratory animal such as a mouse, rabbit or guinea pig, or a whole blood sample derived from a livestock such as a cow, horse, pig or bird. There may be. Further, it may be a whole blood sample immediately after blood collection or a whole blood sample after storage. When using a whole blood sample after storage, the storage conditions are not particularly limited, and it may be stored at room temperature, or may be stored refrigerated or frozen.

The whole blood sample pretreatment method of the present invention comprises (a) a step of diluting a whole blood sample with a diluent solvent containing a polyvalent cation, and (b) a step of heat-treating the diluted whole blood sample. It is characterized by having. Thus, by adding a polyvalent cation to a whole blood sample and heat-treating it, nucleic acids contained in leukocytes are extracted into the solution due to rupture of leukocytes, etc., and impurities such as proteins are removed. It can be inactivated efficiently. This is presumably because the protein denatured by the heat treatment and the polyvalent cation cause a chelate reaction, causing aggregation. In other words, unlike the boiling method in which the sample is boiled to destroy cells and denature proteins, and nucleic acids are extracted, proteins are aggregated by chelation reaction, so that impurities such as lipids and saccharides are also involved. As a result of the formation of aggregates, it is presumed that impurities can be inactivated more effectively.
Hereinafter, it demonstrates for every process.

  First, as step (a), a whole blood sample is diluted with a diluting solvent containing a polyvalent cation. Here, when the dilution rate is too small, the finally obtained treatment solution is almost completely filled with the formed aggregate, and it becomes difficult to collect a sufficient amount of treatment solution for use in the nucleic acid amplification reaction. There is a fear. On the other hand, when the dilution rate is too high, the nucleic acid concentration in the obtained treatment solution may be too low. If the nucleic acid concentration is too low, the amount of the treatment solution supplied to the reaction solution for the nucleic acid amplification reaction increases, and as a result, the reaction scale of the nucleic acid amplification reaction increases and the cost may increase. When the dilution factor is 2 to 100 times, preferably 4 to 64 times, a treatment solution preferable for the nucleic acid amplification reaction can be recovered from both the amount and the nucleic acid concentration. Furthermore, if the dilution ratio is 16 to 32 times, the amount of precipitation due to inactivated impurities can be sufficiently reduced, and when the obtained treatment solution is subjected to a nucleic acid amplification reaction, Bringing in insoluble materials can be suppressed. For this reason, when performing a series of processes from the pretreatment to the nucleic acid amplification reaction manually using an automated apparatus, the dilution factor is preferably 16 to 32 times.

  The polyvalent cation contained in the dilution solvent used in the step (a) is not particularly limited as long as it does not cause precipitation or the like when brought into the reaction solution of the nucleic acid amplification reaction provided with the treatment solution. Although not intended, it is preferably a divalent or trivalent cation. Moreover, a metal ion may be sufficient and nonmetallic ions, such as barium ion, may be sufficient. From the viewpoint of drainage treatment, polyvalent cations other than heavy metal ions are preferable. In the present invention, the polyvalent cation is preferably an alkaline earth metal ion, an aluminum ion, or an iron ion, and more preferably a magnesium ion, an aluminum ion, or a trivalent iron ion. This is because contaminants in the whole blood sample can be inactivated more effectively.

  The concentration of the polyvalent cation contained in the dilution solvent is sufficient to inactivate contaminants contained in the whole blood sample, and does not inhibit the nucleic acid amplification reaction to which the treatment solution is provided. The concentration is not particularly limited, considering the type of polyvalent cation, the type of dilution solvent, the dilution factor of the whole blood sample, the amount brought into the nucleic acid amplification reaction, the type of nucleic acid amplification reaction, etc. Can be determined as appropriate. Generally, the higher the concentration of the polyvalent cation contained in the dilution solvent, the better the efficiency of the chelation reaction. For this reason, for example, when the dilution ratio is 10 times or more, if the polyvalent cation concentration of the dilution solvent is 1 μM or more, impurities in the whole blood sample can be sufficiently inactivated.

  The upper limit of the polyvalent cation concentration of the diluent solvent is not particularly limited when the polyvalent cation used is a kind of ion that does not particularly affect the nucleic acid amplification reaction such as aluminum ion. . For example, when aluminum ions are used as the polyvalent cation, the concentration of aluminum ions in the diluent solvent is preferably 1 μM or more, more preferably 1 μM to 50 mM, and even more preferably 25 μM to 20 mM. . On the other hand, when a divalent metal ion such as a divalent iron ion is used as the polyvalent cation, it is preferably 1 μM to 2.5 mM, and more preferably 1 μM to 0.5 mM. When trivalent iron ions are used as the polyvalent cation, the concentration is preferably 1 μM to 5 mM, more preferably 1 μM to 2.5 mM, and preferably 0.5 to 2.5 mM. Further preferred.

  On the other hand, when the polyvalent cation is an ion that may affect the nucleic acid amplification reaction such as magnesium ion, the influence on the nucleic acid amplification reaction is considered in consideration of the amount brought into the nucleic acid amplification reaction. The concentration range in which a chelate reaction can be efficiently generated can be appropriately determined while suppressing the above. If the polyvalent cation concentration in the diluent solvent is too high, the nucleic acid amplification reaction may be inhibited. Conversely, if it is too low, the chelation reaction efficiency is low, the inactivation efficiency of contaminants is reduced, and the nucleic acid amplification reaction is not performed. It is because it is inhibited. For example, the polyvalent cation of the diluting solvent is magnesium ion, the dilution rate is 10 times or more, and the amount of treatment solution brought into the nucleic acid amplification reaction is 1/10 or less of the final volume of the reaction solution of the nucleic acid amplification reaction If the concentration is about 1 to 0.5 mM, preferably 25 to 125 μM, contaminants can be efficiently inactivated while suppressing the influence on the nucleic acid amplification reaction.

  The type of the dilution solvent used in the step (a) is not particularly limited as long as it does not inhibit the nucleic acid amplification reaction to which the treatment solution is provided. Preparation of water or a biological sample or nucleic acid analysis is generally possible. For example, a buffer to which a polyvalent cation is added can be used. Examples of the buffer include Tris buffer, phosphate buffer, and citrate buffer. Further, a surfactant, an organic solvent, or the like may be added to the dilution solvent as long as the nucleic acid amplification reaction to which the treatment solution is provided is not inhibited.

  Thereafter, as step (b), the whole blood sample diluted in step (a) (diluted whole blood sample) is heat-treated. The temperature of the heat treatment is not particularly limited as long as it is a temperature that can inactivate impurities in the whole blood sample, but it is preferably not more than boiling treatment (100 ° C.). In particular, in order to inactivate impurities by a chelate reaction, it is more preferable that the heat treatment is performed at a relatively lower temperature than the boiling treatment (100 ° C.). For example, in the whole blood sample pretreatment method of the present invention, the heat treatment temperature is preferably 50 to 100 ° C, more preferably 50 to 90 ° C, and even more preferably 65 to 90 ° C. 80 to 90 ° C. is particularly preferable.

  The heat treatment time can be appropriately determined in consideration of the temperature of the heat treatment, the amount of diluted whole blood sample, and the like. When the heat treatment temperature is relatively high such as 80 to 90 ° C., a shorter treatment time may be used than when the heat treatment temperature is relatively low such as 50 to 70 ° C. For example, when the heat treatment temperature is 80 ° C. or higher, the heat treatment time is preferably within 10 minutes, more preferably within 2.5 minutes, and even more preferably within 1 minute. The time for the heat treatment includes not only the time for holding the diluted whole blood sample at the predetermined heat treatment temperature but also the time for the heating step. This is because the inactivation of impurities proceeds even in this heating step. For example, when the heat treatment of step (b) is performed at 80 ° C. or higher after performing step (a) at room temperature, the step of heating to 80 ° C. generally requires several seconds to several tens of seconds. For this reason, the heat treatment may be terminated when the temperature of the diluted whole blood sample reaches 80 ° C.

  By the heat treatment, impurities in the whole blood sample are inactivated and aggregated. Aggregates obtained by this aggregation are much stronger than aggregates due to proteins denatured by boiling, so that, for example, when centrifuged, the volume of the precipitate is reduced and more You can get Qing.

  Since the treatment liquid obtained by treating the whole blood sample by the whole blood sample pretreatment method of the present invention is a nucleic acid extract in which impurities are inactivated, the whole blood sample pretreatment method of the present invention is It is suitable as a pretreatment for nucleic acid analysis using a whole blood sample. In addition, since the nucleic acid amplification reaction can be performed satisfactorily even if inactivated impurities in the treatment liquid are brought into the reaction solution of the nucleic acid amplification reaction, the pretreatment method of the whole blood sample of the present invention Is particularly suitable as a pretreatment method for nucleic acid analysis using a nucleic acid amplification reaction. Although it is not clear why the inactivated contaminants (aggregates) in the treatment solution obtained by the pretreatment method of the whole blood sample of the present invention do not inhibit the nucleic acid amplification reaction, This is presumably because it is very strong compared to aggregates of proteins denatured by boiling treatment.

  The treatment solution obtained by the whole blood sample pretreatment method of the present invention can be used for nucleic acid analysis such as nucleic acid amplification reaction, as with other nucleic acid samples. Examples of the nucleic acid amplification reaction to which the treatment solution is provided include, for example, PCR (polymerase chain reaction) method, LCR (ligase chain reaction) method, SDA (strand displacement amplification) method, LAMP (Loop-Mediated Isotropic IC) method. (Issomer and Chimeric primer-initiated Amplification of Nucleic Acids) method. In the present invention, the PCR method is particularly preferable. These techniques can be carried out under general reaction conditions except that the treatment solution obtained by the pretreatment method of the whole blood sample of the present invention is used as the nucleic acid.

  As described above, inactivated contaminants in the treatment solution hardly inhibit the nucleic acid amplification reaction. Therefore, the treatment solution can be added to the reaction solution of these nucleic acid amplification reactions without worrying about the introduction of aggregates due to contaminants. Further, the aggregate obtained by the chelate reaction has a small volume due to its strong cohesive force and is likely to precipitate. For this reason, for example, from the viewpoint of analysis operation after the nucleic acid amplification reaction, etc., when it is desired to suppress the introduction of aggregates, after the heat treatment in the step (b), by lowering the liquid temperature to about room temperature, By adding the treatment liquid collected from the upper layer portion of this treatment liquid to the reaction solution by precipitating the aggregates, the amount of aggregate brought in can be easily reduced.

  In addition, for example, an aggregate obtained when using a trivalent cation such as aluminum ion or trivalent iron ion is an aggregate obtained when using a divalent cation such as magnesium ion or divalent iron ion. The inhibitory effect on the nucleic acid amplification reaction is smaller. For this reason, the treatment after the heat treatment is easier when the trivalent cation is used than when the divalent cation is used without considering the amount of impurities brought into the reaction solution of the nucleic acid amplification reaction. The solution can be subjected to a nucleic acid amplification reaction. This is presumably because the higher the valence of the polyvalent cations, the higher the flocculation ability and the ability to inactivate impurities efficiently.

  Thus, the pretreatment method of the whole blood sample of the present invention is different from the conventional pretreatment method, even when the step of removing aggregates from the obtained treatment liquid by centrifugation is omitted, A treatment solution suitable for nucleic acid analysis can be obtained. Therefore, the whole blood sample pretreatment method of the present invention can easily automate the whole process using an apparatus including a solution addition unit and a temperature control unit without providing a complicated centrifugation processing unit. is there. In addition, a series of steps from the pretreatment step to the nucleic acid amplification reaction are fully automated by a simple configuration device equipped with a solution dispensing device in a nucleic acid amplification reaction device equipped with a temperature control unit such as a commercially available PCR device. It is also easy.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
Using magnesium ions with different concentrations as the polyvalent cation, whole blood collected from humans by the pretreatment method for whole blood samples of the present invention is pretreated, PCR is performed, and an amplification product of the gene to be analyzed is obtained. Obtained. The subunit 1 (VKORC1) gene of human vitamin K epoxide reductase complex (VKORC) and the human liver cytochrome P450 (cytochrome P450: CYP) 2C9 gene were analyzed.
First, 5 μL of whole blood was diluted with 155 μL of a bittern solution (magnesium sulfate, manufactured by Wako Pure Chemical Industries, Ltd.) having a different magnesium sulfate concentration (dilution ratio is 32 times) and mixed. The magnesium sulfate concentration in the bittern liquid was 1.88 mM to 7.3 μM. These diluted solutions were subjected to heat treatment at 70 ° C. for 5 minutes with a thermal cycler, and then set to 25 ° C. to obtain treatment solutions.
Using these treatment solutions as templates, multiplex PCR was performed using the primers shown in Table 1. By using these primers, an amplification product of 82 bp CYP2C9 gene and an amplification product of 144 bp VKORC1 gene can be obtained, respectively.

Specifically, in a 2 μL treatment solution, the final concentration is 1 × PCR Buffer (manufactured by Toyobo Co., Ltd.), 0.2 mM dNTP, 1 mM magnesium sulfate, 10 μL human genome specimen (20 ng / μL), 0.3 μM. Each primer and 0.9 μL of KODplus (manufactured by Toyobo Co., Ltd.) were added, and milliQ water was added to prepare a total solution of 36 μL. In addition, it replaces with a process liquid, 10 ng of human-extracted genomes confirmed that the amplification product of VKORC1 gene and CYP2C9 gene is obtained using the primer of Table 1 (provided by Human Science Foundation) Those obtained using the Human Science Training Resource Bank in an unanonymized state) were used as PCR positive controls, and those using an equal amount of milliQ water were used as negative controls.
These reaction solutions were subjected to a denaturation step at 94 ° C. for 5 minutes, followed by 35 cycles of amplification steps at 94 ° C. for 30 seconds, 64 ° C. for 30 seconds, 68 ° C. for 30 seconds, 68 ° C. for 2 minutes, PCR reaction was performed under the reaction conditions consisting of Whether the target CYP2C9 amplification product (82 bp) and VKORC1 amplification product (144 bp) are amplified by electrophoresis of the reaction solution after the PCR reaction using 8% polyacrylamide gel and then staining with SYBR Green1 It was confirmed.

  FIG. 1 is a staining diagram showing a band pattern obtained by SYBR Green1 staining. Lane M is a molecular weight marker 20 bp ladder (manufactured by Takara Bio Inc.), and lanes 1 to 9 are reaction solutions using a treatment solution pretreated with a bitumen solution having a magnesium sulfate concentration shown in Table 2 as a template. In Lane 10, positive controls (using human genome as a template) were run. Furthermore, the density of each band was measured with a densitograph, and the results of measuring the amount of each amplification product are shown in FIG.

As a result, both the CYP2C9 amplification product and the VKORC1 amplification product were detected in the lanes 3 to 9 where the concentration of magnesium ions in the bittern solution used for the pretreatment was 7.3 μM to 0.469 mM, as in the lane 10. In particular, in the lanes 5 to 7 where the magnesium ion concentration was 0.029 to 0.117 mM, both amplification products could be obtained in a balanced manner. On the other hand, only the CYP2C9 amplification product was detected in lane 1, and only the VKORC1 amplification product was detected in lane 2.
From these results, it is clear that the treatment liquid treated by the whole blood sample pretreatment method of the present invention can be subjected to a nucleic acid amplification reaction without performing a centrifugation treatment. In general, a liquid containing magnesium ions is not used for pretreatment for a nucleic acid amplification reaction using a polymerase such as PCR. This is because it is well known that the magnesium ion concentration in the polymerase reaction has an optimum value, and it is necessary to avoid bringing in magnesium from the specimen. Nevertheless, in the present invention, even when the magnesium ion-containing liquid was used, the pretreatment could be performed satisfactorily.

[Example 2]
The effect of heat treatment temperature in pretreatment was investigated.
Specifically, the whole blood sample was pretreated in the same manner as in Example 1 except that the magnesium sulfate concentration of the bittern liquid used for the pretreatment was 0.117 mM and the temperature of the heat treatment was 50 ° C to 90 ° C. Then, a PCR reaction was performed using the pretreatment solution as a template, and the obtained amplification product was detected by SYBR Green 1 staining after electrophoresis.
FIG. 3 is a staining diagram showing a band pattern obtained by SYBR Green1 staining. Lane M is a molecular weight marker 20 bp ladder (manufactured by Takara Bio Inc.), lanes 1 to 5 are reaction solutions prepared using a treatment solution pretreated at the heat treatment temperature shown in Table 3, and lane 6 is a positive control. (Using human genome as a template). Furthermore, the density of each band was measured with a densitograph, and the results of measuring the amount of each amplification product are shown in FIG.

  As a result, in all lanes, both the CYP2C9 amplification product and the VKORC1 amplification product were detected with sufficient intensity, and multiplex amplification was achieved in both. In particular, when the heat treatment temperature in the pretreatment was 80 ° C. to 90 ° C., nonspecific amplification could be reduced. Since the signal intensity was high and non-specific amplification was small, the heat treatment temperature was most suitable at 80 ° C. in this example.

[Example 3]
The effect of heat treatment time on pretreatment was investigated.
Specifically, except that the magnesium sulfate concentration of the bittern liquid used for the pretreatment is 0.117 mM, the temperature of the heat treatment is 80 ° C., and the heat holding time after reaching 80 ° C. is 0 to 10 minutes, In the same manner as in Example 1, a whole blood sample was pretreated, a PCR reaction was performed using the pretreated solution as a template, and the obtained amplification product was detected by SYBR Green1 staining after electrophoresis.
FIG. 5 is a staining diagram showing a band pattern obtained by SYBR Green1 staining. Lane M is a molecular weight marker 20 bp ladder (manufactured by Takara Bio Inc.), lanes 1 to 5 are positive with a reaction solution pretreated with the heating and holding time shown in Table 4 as a template, and lane 6 is positive. Each control (using human genome as a template) was run. Furthermore, the density of each band was measured with a densitograph, and the results of measuring the amount of each amplification product are shown in Table 4 and FIG.

  As a result, in all lanes, both the CYP2C9 amplification product and the VKORC1 amplification product were detected with sufficient intensity, and multiplex amplification was achieved in both. In particular, when the heating and holding time in the pretreatment was 0 minutes (when heated to 80 ° C. and immediately brought to 25 ° C.), the largest amount of amplified product was obtained.

[Example 4]
The effect of dilution factor of whole blood on pretreatment was examined.
First, the magnesium sulfate concentration of the bittern solution used for the pretreatment is set to 0.117 mM, the dilution ratio for 5 μL of whole blood is set to 4 to 64 times, the temperature of the heat treatment is set to 80 ° C., and the heat retention after reaching 80 ° C. A whole blood sample was pretreated in the same manner as in Example 1 except that the time was set to 0 minute, PCR reaction was performed using the pretreatment liquid as a template, and the obtained amplification product was stained with SYBR Green1 after staining. Detected by.
FIG. 7 is a staining diagram showing a band pattern obtained by SYBR Green1 staining. Lane M is a molecular weight marker 20 bp ladder (manufactured by Takara Bio Inc.), lanes 1 to 5 are reaction solutions using a pretreated treatment solution at dilution ratios shown in Table 5 as templates, and lane 6 is a positive control. (Using human genome as a template). Furthermore, the density of each band was measured with a densitograph, and the results of measuring the amount of each amplification product are shown in FIG.

As a result, in all lanes, both the CYP2C9 amplification product and the VKORC1 amplification product were detected, and multiplex amplification was achieved in both cases. In particular, in lanes 2 to 5 where the dilution factor was 4 to 32, both amplification products could be detected with sufficient intensity.
On the other hand, FIG. 9 is a diagram observing the state of the treatment liquid obtained after the pretreatment. When the dilution ratio was 4 times or 8 times, a large amount of precipitate (blood denatured product) was generated in the treatment liquid obtained after the pretreatment. For this reason, when applied to an automated apparatus, it is preferable to carry out at a dilution factor of 16 to 32 times with a small amount of precipitation and a sufficient amount of amplification product.

[Example 5]
Using aluminum ions as polyvalent cations, 10 samples of whole blood collected from a human by the whole blood sample pretreatment method of the present invention are pretreated, PCR is performed, and an amplification product of the gene to be analyzed is obtained. It was. The analysis target genes were the VKORC1 gene and the CYP2C9 gene, as in Example 1.
First, 6.25 μL of each whole blood was diluted with 43.75 μL of an aqueous aluminum sulfate solution (aluminum sulfate, manufactured by Wako Pure Chemical Industries, Ltd.) and mixed. The aluminum sulfate concentration in these dilutions was 10 mM. These diluted solutions were subjected to a heat treatment at 80 ° C. for 5 minutes by a thermal cycler and then set to 25 ° C. to obtain a treated solution.
In the same manner as in Example 1, PCR reaction was performed using these treatment solutions as templates, and the obtained amplification product was detected by SYBR Green1 staining after electrophoresis.
FIG. 10 is a staining diagram showing a band pattern obtained by SYBR Green1 staining. Lane M is a molecular weight marker 20 bp ladder (manufactured by Takara Bio Inc.), lanes 1 to 10 are reaction solutions using a treatment solution obtained from 10 samples of whole blood, and lane 11 is a positive control (human genome). Lane 12 and negative control (added with milliQ water) were allowed to flow in lane 12 respectively. As a result, although there was a difference in the amount of amplification depending on the sample, both genes could be detected by multiplex PCR in any sample.

[Example 6]
Using divalent iron ions and trivalent iron ions as the polyvalent cations, the influence of the valence of the polyvalent cations was observed.
Specifically, 6.25 μL of whole blood was mixed with 43.75 μL of ferrous chloride aqueous solution (FeCl ₂ , manufactured by Wako Pure Chemical Industries, Ltd.) or ferric chloride aqueous solution (FeCl ₃ , Wako Pure Chemical Industries, Ltd.) having different concentrations. The product was diluted with 43.75 μL and mixed. The concentration of ferrous chloride or ferric chloride in these dilutions was 0.5-50 mM. These diluted solutions were subjected to a heat treatment at 80 ° C. for 5 minutes by a thermal cycler and then set to 25 ° C. to obtain a treated solution.
In the same manner as in Example 1, PCR reaction was performed using these treatment solutions as templates, and the obtained amplification product was detected by SYBR Green1 staining after electrophoresis.
FIG. 11 is a staining diagram showing a band pattern obtained by SYBR Green1 staining. Lane M is a molecular weight marker 20 bp ladder (manufactured by Takara Bio Inc.), and lanes 1 to 6 are pretreated with a ferrous chloride aqueous solution having a ferrous chloride (FeCl ₂ ) concentration shown in Table 6. The reaction solutions using the solution as a template are shown in lanes 9 to 14 using the treatment solution pretreated with a ferric chloride aqueous solution having a ferric chloride (FeCl ₃ ) concentration shown in Table 7 as a template. Lanes 7 and 15 were fed with a positive control (using human genome as a template), and lanes 8 and 16 were fed with a negative control (added with milliQ water), respectively. As a result, both genes could be detected by multiplex PCR both when using divalent iron ions (FeCl ₂ ) and when using trivalent iron ions (FeCl ₃ ). In particular, a tendency was observed that the amount of amplified product was larger when trivalent iron ions were used than when divalent iron ions were used. When an aqueous ferric chloride solution is used, PCR amplification may be very good when the trivalent iron ion concentration in the diluted solution is 2.5 to 0.5 mM, particularly 2.5 mM. I understood.

  By using the whole blood sample pretreatment method of the present invention, it is possible to easily prepare nucleic acid samples suitable for nucleic acid analysis, particularly nucleic acid amplification reaction, without performing centrifugation treatment. It can be used in fields such as clinical tests where genetic analysis is performed.

In Example 1, it is the dyeing | staining image which showed the band pattern obtained by carrying out polyacrylamide gel electrophoresis of the reaction solution after PCR reaction, and dyeing | staining SYBR Green1. In Example 1, it is the figure which showed the result of having measured the density of each band obtained by SYBR Green1 dyeing | staining with the densitograph. In Example 2, it is the dyeing | staining image which showed the band pattern obtained by carrying out polyacrylamide gel electrophoresis of the reaction solution after PCR reaction, and dyeing | staining SYBR Green1. In Example 2, it is the figure which showed the result of having measured the density of each band obtained by SYBR Green1 dyeing | staining with the densitograph. In Example 3, it is the dyeing | staining image which showed the band pattern obtained by carrying out polyacrylamide gel electrophoresis of the reaction solution after PCR reaction, and dyeing | staining SYBR Green1. In Example 3, it is the figure which showed the result of having measured the density of each band obtained by SYBR Green1 dyeing | staining with the densitograph. In Example 4, it is the dyeing | staining image which showed the band pattern obtained by carrying out polyacrylamide gel electrophoresis of the reaction solution after PCR reaction, and SYBR Green1 dyeing | staining. In Example 4, it is the figure which showed the result of having measured the density of each band obtained by SYBR Green1 dyeing | staining with the densitograph. In Example 4, it is the figure which observed the state of the process liquid obtained after the pre-processing. In Example 5, it is the dyeing | staining image which showed the band pattern obtained by carrying out polyacrylamide gel electrophoresis of the reaction solution after PCR reaction, and dyeing | staining SYBR Green1. In Example 6, it is the dyeing | staining image which showed the band pattern obtained by carrying out polyacrylamide gel electrophoresis of the reaction solution after PCR reaction, and SYBR Green1 dyeing | staining.

Claims (6)

A pretreatment method for amplifying nucleic acid in a whole blood sample, comprising:
(a) diluting a whole blood sample with a diluting solvent containing a polyvalent cation;
(b) heat treating the whole blood sample diluted in step (a);
A pretreatment method for a whole blood sample, comprising:   2. The pretreatment method for a whole blood sample according to claim 1, wherein the polyvalent cation is an alkaline earth metal ion or an aluminum ion.   2. The pretreatment method for a whole blood sample according to claim 1, wherein the polyvalent cation is aluminum ion or iron ion.   The pretreatment method for a whole blood sample according to any one of claims 1 to 3, wherein the temperature of the heat treatment in the step (b) is 50 to 100 ° C.   The pretreatment method of a whole blood sample according to claim 1 or 2, wherein the temperature of the heat treatment in the step (b) is 50 to 90 ° C.   A nucleic acid amplification method comprising performing a nucleic acid amplification reaction using a treatment solution pretreated using the whole blood sample pretreatment method according to claim 1.