JP5521153B2 - Rapid detection method for single nucleotide polymorphisms in blood samples - Google Patents

Description

  The present invention relates to a method for rapid detection of single nucleotide polymorphisms in a blood sample, and relates to the field of clinical diagnosis such as point-of-care testing (POCT), the field of clinical research dealing with blood samples, and the field of differential identification in forensic science.

  In recent years, association risk analysis using single nucleotide polymorphisms (SNPs or SNPs) has identified genetic risk factors for susceptibility and drug responsiveness of various diseases. Along with this, SNP genotyping is performed as a point-of-care test (patient's personal test: POCT) in clinical practice, and the movement to realize individual medical care is accelerating.

  The results of the Human Genome Sequencing Project and the International HapMap Project have made it possible to identify genetic risk factors by association analysis using SNPs, and innovative individual medicine is becoming a reality. Knowledge regarding the correlation between SNPs and clinically important phenotypes (ease of disease, disease occurrence, drug response, etc.) has been rapidly accumulated (Non-Patent Documents 1 to 5). Some of these are close to clinical application (Non-Patent Documents 6 and 7). For example, the drug response of “Warfarin”, the most commonly used oral anticoagulant for the treatment of thromboembolism, is associated with several SNPs of CYP2C9 * 2, CYP2C9 * 3 and VKORC1 It has been reported that the optimal dosage in warfarin sensitive or insensitive patients can be predicted to some extent from the genotyping results of SNPs (Non-patent Documents 2 and 3). The warfarin package insert has already been revised and it is recommended to refer to the genotyping data of SNPs at the time of prescribing. The FDA strongly supports the realization of individual medicine based on SNP genotyping that maximizes drug efficacy and minimizes drug toxicity (Non-Patent Document 8). Thus, the demand for SNP genotyping is increasing mainly in the clinical diagnostic field, and it is important to develop an accurate, quick, simple and inexpensive SNP genotyping system from biological samples (Non-patent literature). 9-11).

  Most existing SNP genotyping techniques require nucleic acid amplification by PCR and require a purification step of genomic DNA from biological samples. This is because a biological sample such as whole blood and oral swab (cotton swab specimen) contains a PCR inhibitor such as heme (Non-patent Documents 12 and 13). The traditional purification process of genomic DNA is complex and takes almost half a day, and this process is the biggest obstacle in genotyping for POCT (Non-Patent Documents 9, 11, 14, and 15). In recent years, efforts have been made to simplify the genomic DNA purification process, and useful methods using magnetic beads (Non-Patent Document 16), spin columns (Non-Patent Document 17) or other techniques have been developed ( Non-patent document 18). Several kits or automated systems using the above technology become available (Non-Patent Documents 10 and 18), and it is possible to obtain purified genomic DNA in about 10 to 30 minutes from the collection of biological samples. However, although these are very expensive and simplified as a kit, they still require multiple steps and are not considered suitable for POCT.

  As another approach for solving the obstacle, a pretreatment method such as alkali dissolution or formamide extraction (Non-patent Documents 9, 11, 14, 15) or a promoter such as bovine serum albumin (BSA), betaine and gp32 is used. Depending on the method used (Non-Patent Documents 19 and 20), several methods have been reported that allow PCR or PCR-based reactions without a genomic DNA purification step. Among these, the alkaline lysis method can be applied to various biological samples including whole blood, oral swabs and other biological samples (Non-patent Documents 14 and 15). Incubation under strong alkaline conditions disrupts cell and nuclear membranes, denatures nucleases, neutralizes certain PCR inhibitors, and keeps genomic DNA very stable under strong alkaline conditions (Non-patent documents 21, 22). Furthermore, the alkali dissolution method is very simple and inexpensive. This is a work process in which a biological sample is mixed with a NaOH solution at room temperature before PCR and then neutralized with a Tris-HCl buffer (Non-Patent Documents 14 and 15). Therefore, this method may be a useful genome extraction method for clinical application of SNP genotyping.

  Regarding the SNP genotyping technology, many genotyping methods have been reported so far. For example, melting curve analysis using a hybridization probe (Non-patent document 23), TaqMan method (Non-patent document 24), invader method (Non-patent document 25), molecular beacon (Non-patent document 26) Scorpion primer (Non-patent document 27) ), Array hybridization (non-patent document 28), fluorescence polarization (non-patent document 29), pyrosequencing (non-patent document 30), DNA sequencer (non-patent document 31) or MALDI-TOF mass spectrometry ( Non-patent document 32) Primer extension, oligonucleotide ligation PCR reaction (OLA-PCR), smart amplification process method (non-patent document 11), etc. Many of these techniques have multi-step protocols and require several hours to complete the entire reaction. In POCT, it is necessary to be a simplified protocol, to be a single tube assay format, and to shorten the reaction time within 1 hour (Non-patent Document 10). At present, melting curve analysis and TaqMan method are known as methods that generally satisfy these conditions, and are widely used in the clinical field. Recently, it has been reported that whole blood samples treated with formamide can be used in these techniques (Non-patent Document 9), and the reaction time was about 40 minutes by melting curve analysis and about 2 hours by TaqMan method. Thus, the method may be a promising candidate for POCT, but is expensive due to the need for target-specific fluorescent probes compared to others, and reported to have a low signal-to-noise ratio compared to other assays. (Non-Patent Documents 11, 27, 33).

The Invader (registered trademark) method is known as one of the most reliable SNP genotyping technologies and has a track record of being used in the International HapMap project (Non-patent Document 34). . In the invader method, a specific triple structure formed by two types of oligonucleotide probes at the target site of the nucleotide sequence is flap. It utilizes that enzyme called endonuclease (Cleavase (registered trademark) VIII) recognizes and cleaves. A part of the cleaved DNA fragment (Flap sequence) reacts with a universal fluorescence resonance energy transfer (FRET) probe (Non-patent Document 35) to emit fluorescence, so that the genotype at the target SNP site can be determined. . Since the generation of a fluorescent signal in the invader method requires the formation of a specific triple structure by the oligonucleotide probe, the invader method shows a higher target specificity than the method by hybridization alone. Moreover, since two types of universal FRET probes can be used for all target SNP assays, they are more cost-effective than melting curve methods and TaqMan methods that require a fluorescent probe for each target SNP (Non-patent Document 33). In the related analysis using SNP, the invader method is performed by a two-step reaction protocol by combining with PCR that simultaneously amplifies 96 SNP sites (Non-patent Document 35). The two-step protocol achieved high-throughput SNP genotyping with a very small amount of genomic DNA (0.1 ng / locus), but because it is not a single tube format, there is a risk of contamination in the post-PCR process, and the reaction time is also long. There was a drawback that it took several hours (Non-patent Document 33). The Universal Invader Method (http://www.universalinvader.com/twt/uic/) was developed to overcome these drawbacks and enable clinical applications, and was developed by Third Wave Technologies (Madison, WI). It was announced. This method is a combination of PCR and Invader® reaction, in which PCR and invader reactions proceed in a continuous manner in a single tube assay format. The principle of the universal invader method is shown in FIG. The universal invader method has a simplified protocol that minimizes contamination risk and can be expected to respond quickly (http://www.universalinvader.com/twt/rh.pdf).
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  An object of the present invention is to provide an inexpensive SNP genotyping method that is used quickly and simply as a point-of-care test.

The biggest obstacles to realizing the above problems are the purification process of genomic DNA from biological samples and the long reaction time in SNP genotyping technology. The present inventors have combined a modified alkaline lysis method and a universal invader method to provide a simple method for preparing genomic DNA that does not require purification of a blood sample and SNP using the prepared genomic DNA as a test sample as it is. The genotyping method was successfully established and the present invention was completed.
That is, the present invention is as follows.

[1] Method for preparing single nucleotide polymorphism test sample for genotyping including the following steps:
Mixing 4 to 40 parts by volume of an aqueous solution of 100 to 200 mM alkali metal hydroxide with 1 part by volume of an animal-derived blood sample;
A step of adding 0.5 to 5 volumes of 80 to 560 mM Tris buffer to the mixed solution to neutralize, and a step of centrifuging or filtering the neutralized solution to obtain a test sample;
[2] Single nucleotide polymorphism genotyping inspection method including the following steps:
Mixing 4 to 40 parts by volume of an aqueous solution of 100 to 200 mM alkali metal hydroxide with 1 part by volume of an animal-derived blood sample;
Adding 0.5 to 5 volumes of 80 to 560 mM Tris buffer to the mixed solution, and neutralizing;
Centrifuge or filter the neutralized solution to obtain a test sample, and genotyping by the universal invader method using the test sample.
[3] The method according to [1] or [2] above, wherein the alkali metal hydroxide is sodium hydroxide or potassium hydroxide.
[4] The method according to [1] or [2] above, wherein the Tris buffer is Tris-HCl (pH 7 to 9, 25 ° C.).
[5] The method according to any one of [1] to [4], wherein the process from the mixing step to the step of obtaining a test sample is performed within 5 minutes.
[6] The method according to any one of [2] to [5], wherein the universal invader method is performed in the presence of 4.5 to 9.0 mM MgCl ₂ and 0 to 20 mM NaCl.
[7] The method according to any one of [2] to [6], wherein the mixing step to the genotyping step are performed within 30 minutes.
[8] A kit for preparing a single nucleotide polymorphism test sample for genotyping, comprising an aqueous solution of 100 to 200 mM alkali metal hydroxide and an 80 to 560 mM Tris buffer.
[9] The kit according to [8], further including a mixing container and / or a centrifuge tube.

  The preparation method of the present invention is a blood sample processing method for performing the universal invader method accurately and quickly, and is useful as a pretreatment method for performing genotyping from a blood sample conveniently, quickly and accurately in clinical practice. is there. The test method of the present invention uses the test sample obtained by the preparation method of the present invention and is accurate, simple and inexpensive, and can obtain a genotyping result from a blood sample within 30 minutes. This feature suggests that the method of the present invention is one of the most promising methods for realizing SNP genotyping as POCT. In addition, the kit of the present invention is useful for carrying out the method of the present invention accurately and rapidly in the medical field.

  The present invention provides a method for preparing a single nucleotide polymorphism test sample for genotyping, comprising the following steps (1) to (3).

(1) A step of mixing 4 to 40 parts by volume of an aqueous solution of 100 to 200 mM alkali metal hydroxide with 1 part by volume of an animal-derived blood sample.

  In the present invention, the animal is not particularly limited and includes invertebrates and vertebrates, with vertebrates being preferred. Vertebrate animals include fish, amphibians, reptiles, birds and mammals. Among them, mammals (eg, laboratory animals such as mice, rats, and rabbits, pets such as cats and dogs, domestic animals such as cows and horses, primates such as monkeys and humans) are more preferable as test targets, most preferably humans. Including primates.

  In the present invention, a blood sample refers to a liquid sample that contains various blood cell components according to the type of animal and circulates in the living body of the animal. Examples of blood include whole blood, plasma, serum, blood clot, etc. Whole blood is preferable from the viewpoint of obtaining genome information comprehensively without requiring pretreatment after collection. When blood is collected from an animal, an anticoagulant may or may not be mixed. Therefore, in the present invention, whole blood may be coagulated. In addition, the blood sample is not limited to one collected directly from the animal to be examined, and blood once released from the animal outside the body may be collected outside the purpose of collection (for example, due to bleeding or the like).

  A method for collecting a blood sample from the animal to be examined follows a conventional method. The amount to be collected is not particularly limited, but 1 to 10 μL is usually sufficient.

  The collected blood sample may be immediately subjected to the preparation method of the present invention, or may be subjected to refrigeration or frozen storage according to a conventional method.

  In this step, 4 to 40 parts by volume of an aqueous solution of 100 to 200 mM alkali metal hydroxide is mixed with 1 part by volume of the collected blood sample.

  The alkali metal hydroxide to be mixed is preferably sodium hydroxide or potassium hydroxide, but is not limited thereto.

  The concentration of the alkali metal hydroxide aqueous solution is 100 to 200 mM, preferably 200 mM. By mixing with an alkali metal hydroxide having a concentration within such a range, the cell membrane and the nuclear membrane in the blood sample are destroyed, the nuclease is denatured, and the lysis of the genomic DNA in the aqueous solution is achieved.

  In order to achieve dissolution of the genomic DNA, the mixing ratio of the blood sample and the aqueous solution of alkali metal hydroxide is 1 to 50 parts by volume, preferably 4 to 40 parts by volume, more preferably 4 to 20 parts by volume. The higher the alkali metal hydroxide concentration, the lower the mixing ratio. In general, when performed at a clinical site, 10 to 50 μL of a 200 mM alkali metal hydroxide aqueous solution is recommended for 1 to 10 μL of a blood sample.

  The mixing step is usually completed by stirring or standing at 10 to 30 ° C. for 1 to 2 minutes.

(2) A step of adding 0.5 to 5 volumes of 80 to 560 mM Tris buffer to the mixed solution to neutralize it

  The Tris buffer used in the neutralization step is not particularly limited as long as it can neutralize the alkali metal hydroxide aqueous solution used in step (1). The commonly used Tris-HCl is preferred. The pH of a Tris buffer solution typified by Tris-HCl is preferably in the range where the buffering ability of Tris is exhibited, and is usually pH 7 to 9 (25 ° C.).

  In this neutralization step, the concentration of the Tris buffer is set to 80 to 560 mM, preferably 160 to 240 mM in order to precipitate and remove components in the blood sample that inhibit the subsequent PCR reaction.

The addition ratio of Tris buffer to the mixed solution obtained in the mixing step is 0.4 to 10 times the volume of Tris buffer with respect to the total volume of the mixed solution in order to achieve the intended purpose of the present invention. The volume is preferably 0.5 to 5 times, and more preferably 1 to 2 times. The higher the Tris buffer concentration, the lower the addition ratio. In general, when carried out at a clinical site, 20 to 100 μL of 160 mM Tris buffer is recommended for 10 to 50 μL of the mixed solution.
The entire solution may be stirred gently so that neutralization is complete after the addition.

(3) Centrifugation or filtration of the neutralized solution to obtain a test sample Through the neutralization step, protein components and the like in the blood sample are generated as precipitates. By this step, the precipitate is removed from the blood sample.

  Centrifugation conditions are not particularly limited as long as the precipitate and the supernatant can be separated, and are usually exemplified by 0.5 to 1 minute at 1000 to 3000 rpm. In clinical settings, centrifugation can be performed using a desktop centrifuge. Moreover, it is also possible to remove the precipitate with a filter instead of centrifugation.

  In order to use for centrifugation at this process, it is preferable that process (1)-(2) is performed within the container which can perform both mixing operation and centrifugation operation. Thereby, steps (1) to (3) can be performed in the same container. Moreover, it is also possible to remove the precipitate by filtration using a container with a filter instead of centrifugation. The containers that can be used in the method of the present application will be described later.

  In the preparation method of the present invention, the steps (1) to (3) can be carried out within 5 minutes. As a result, blood samples can be rapidly pretreated even in clinical settings.

  The present invention provides a kit for preparing a single nucleotide polymorphism test sample for genotyping, which allows pretreatment of a specimen (blood sample) collected from a subject at the clinical site in the preparation method of the present invention. The kit of the present invention comprises an aqueous solution of 100-200 mM alkali metal hydroxide and 80-560 mM Tris buffer.

  The kit of the present invention may further include a mixing container and / or a centrifuge tube that can be suitably used for pretreatment of a blood sample. As such a mixing container and / or centrifuge tube, a container conventionally used for nucleic acid extraction can be used without limitation. Preferably, with a sampling tube with a lid made of propylene (eg, Eppendorf, 0.5, 1.5, 2.0, 5.0 ml capacity, etc.) or syringe (Terumo 0.5, 1.0, 2.0, 5.0 ml capacity, etc.), blood and alkaline solution, And a container capable of easily mixing the mixed solution and the Tris buffer. In addition, a container to which a filter (0.45 μm pore size, etc.) can be attached can be used to remove precipitates. Furthermore, a container used in this technical field can be used without limitation when genotyping of single nucleotide polymorphism is performed automatically from sampling of a blood sample.

  The present invention provides a single nucleotide polymorphism genotyping test method comprising the following step (4) in addition to the steps (1) to (3).

(4) A step of genotyping by the universal invader method using the test sample obtained in the step (3).

  The principle of the universal invader method used in the present invention is shown in FIG. The universal invader method is a measurement method in which PCR and invader reaction are continuously performed in a single tube format. Five unlabeled oligonucleotides (forward primer, reverse primer, invader probe and two allele probes), dNTP, two FRET probes, Taq polymerase and Cleavase VIII are in the same reaction mixture. Of the five oligonucleotides, only the PCR primer works at the PCR stage, and the oligonucleotide related to the invader method cannot react at the PCR stage (annealing / extension, 68 ° C.) because of its low Tm value. After PCR, the invader reaction starts continuously. The invader method is a method that identifies two different alleles of a SNP in a single tube. For each target SNP, two allele probes complementary to each allele are designed. Each allelic probe has a different universal flap sequence at the 5 'end. In addition, one invader probe for each target SNP is designed at the site opposite the allele probe. In the primary invader reaction, when the invader probe and the allele probe hybridize to complementary target DNA, a single base invasion structure (triple structure) by the invader probe is formed at the SNP site, and Cleavase VIII that recognizes the specific structure is the allele. Cleave the target SNP site of the probe. The arrow indicates the cleavage site. Cleaved oligonucleotides with bases at the Flap and SNP sites hybridize with a specific fluorescence resonance energy transfer (FRET) probe having a sequence complementary to the flap sequence and behave as an invader probe in a secondary invader reaction. Cleavase VIII recognizes the invasion structure and cleaves the FRET probe, generating a fluorescent signal. The FRET probe uses two different reporter fluorescent dyes and is designed to be complementary to each flap sequence. The primary and secondary invader reactions proceed continuously. If the probe does not completely hybridize at the site of interest and no invasion structure is formed, no cleavage occurs and no fluorescent signal is generated. SNP genotyping is usually done by clustering analysis in a two-dimensional allele discrimination (AD) plot at the end of the reaction.

  The five unlabeled oligonucleotides (forward primer, reverse primer, invader probe, and two allele probes (allele 1 probe, allele 2 probe)) used in the universal invader method are universal invader design software (Third Wave Technologies, http://www.universalinvader.com/twt/uic/). However, since a blast search or the like is not performed, when constructing an assay for a target having a similar sequence in the genome such as a drug metabolizing enzyme having a gene family as in this example, a manual considering the Tm value Primer design is required. Also, if the reaction is poor with the above design software, manual primer and probe design is required. Specific examples of oligonucleotides used in Examples of the present application are shown in Table 1 and SEQ ID NOs: 1-283.

  A part of the test sample (supernatant) obtained in the step (3) can be directly added to a reaction solution for the universal invader method to carry out a universal invader reaction.

In this step (4), it is important to strictly control the MgCl ₂ concentration and the NaCl concentration in the reaction solution in order to allow the PCR and invader reaction to proceed well. The MgCl ₂ concentration is preferably 4.5 to 10.5 mM, more preferably 6.0 to 7.5 mM. The NaCl concentration is preferably 0 to 20 mM, more preferably 0 to 10 mM. The composition in the reaction solution other than MgCl ₂ and NaCl is 10-20 mM Tris buffer (pH about 8.0-8.8) or MOPS buffer (pH 7.0-8.0), 0-5% glycerol, 0.8-1.6 mM deoxyribo. Nucleotide triphosphates (dNTPs), 0.2-2.0 μM forward and reverse primers, 0.1-0.4 μM invader probe, 0.2-0.8 μM allele probe, 0.1-0.5 μM FRET probe, 10-40 μM Rox, 20-100 ng Cleavase VIII 0.1-0.5U Taq polymerase etc. are illustrated.

  The total reaction time of the step (4) is usually within 25 minutes, and the steps (1) to (4) can be completed within 30 minutes.

  In the SNP genotyping in step (4), two types of fluorescence signals emitted from the cleaved FRET probe were measured with an Applied Biosystems 7500 real-time PCR system, an Applied Biosystems 7900 real-time PCR system, a Roche Lightcycler 480, etc. A dimensional allele identification (AD) plot is created and performed in a clustering analysis.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

1. Samples tested 18 whole blood samples were collected from Japanese healthy volunteers in the laboratory to which the inventors belonged. The collected whole blood samples were treated with the anticoagulant EDTA and stored at −80 ° C. until use. To confirm the results of SNP genotyping from whole blood samples, QIAamp® DNA Blood MiniKit (Qiagen, Hildden, Germany) was used to obtain a purified genomic DNA sample according to the manufacturer's recommended protocol . Laboratory approval for conducting this study using human material was obtained from the RIKEN Ethics Advisory Committee. In order to confirm the performance of the assay by each universal invader method, the inventors of the present invention used 268 genomic DNA samples (44 Japanese origins, 44 Chinese origins, 90 people) adopted in the international HapMap project. White and 90 Yoruba) were also used. These genomic DNA samples were purchased from Coriell Cell Repositories (Camden, NJ). For assays in which no variant was detected in 268 HapMap samples, an artificial oligonucleotide template covering the amplicon region was synthesized, and it was confirmed whether the assay was effective for both alleles for each polymorphism. The sequence of the artificial template is shown in Table 1.

2. In order to achieve rapid SNP genotyping of whole blood samples from alkaline lysed whole blood samples, we adopted the previously published alkaline lysis method (comparative example) in the early stages of this study [Rudbeck et al, Biotechniques 25: 588-592 (1998)]. Briefly, 5 μl of whole blood sample was mixed with 20 μl of 200 mM NaOH and incubated for 1-2 minutes at room temperature. Thereafter, the alkali-treated blood was neutralized with 180 μl of 40 mM Tris-HCl pH 7.5, and 2 μl of the treated sample was used in a 10 μl universal invader reaction system. Since the previously reported alkaline lysis method was not suitable for the universal invader method, a modified protocol using concentrated Tris-HCl buffer was developed. The modified protocol (invention) was to neutralize a 5 μl whole blood sample treated with 20 μl 200 mM NaOH for 1-2 minutes at room temperature and mixed with 45 μl 160 mM Tris-HCl pH 7.5. . After centrifuging with a desktop centrifuge system Chibitan (Millipore, Billerica, Mass.) To remove the precipitate, 2 μl of the supernatant was used in a 10 μl universal invader reaction system.

3. Universal Invader Assay Design 38 clinically important single nucleotide polymorphisms (SNPs) or several base insertions / deletions (Indels) were selected from several reports on drug responsiveness and disease susceptibility (Table 1). ). For 36 of these 38 targets, invader and allele probes were designed using Universal Invader Design Software (Third Wave Technologies, http://www.universalinvader.com/twt/uic/) and Third Synthesized according to Wave Technologies instructions. For ABCB1 * 3,2677T>G> A, which is an SNP with three alleles, the Tm value is taken into account, and two assays are manually performed in the form of ABCB1 * 3, 2677T> A and ABCB1 * 3, 2677G> A Designed separately. The Tm value conditions at that time were 58 to 62 ° C. for the invader probe and 50 to 53 ° C. for the allele probe. UGT1A1 * 28 (TA repeated 6 or 7 times) was also synthesized manually. PCR primers were performed using Primer Express software (Applied Biosystems, Foster City, Calif.) So that the Tm value was 68 to 72 ° C. Finally, 39 assays were constructed for the 38 polymorphisms. The sequence of each oligonucleotide is shown in Table 1.

4). Universal Invader Assay A universal invader assay was performed using a reaction solution prepared uniquely. Detailed optimization was performed for the concentrations of MgCl ₂ and NaCl, and the concentrations of the other components were determined with reference to the composition of a normal two-step PCR-invader reaction. The composition of the reaction solution was 20 mM Tris-HCl pH 8.5, 3% glycerol, 1.6 mM deoxynucleotide triphosphates (dNTPs), 1.6 μM forward and reverse primers, 200 nM invader probe, 400 nM allele Probe, 200 nM FRET probe labeled with FAM or Yakima Yellow (Third Wave Technology), 10 μM Rox (Sigma, St. Louis, MO), 60 ng Cleavase VIII (Third Wave Technology) and 0.25 U Takara Ex It consists of Taq (Takara, Shiga, Japan). The reaction was performed in a volume of 10 μl including 2 μl of alkali-treated whole blood sample or 0.5 ng / well of purified genome. Fluorescence detection at the reaction temperature cycle and reaction endpoint was performed in the fast reaction mode of the 7500 First Real-time PCR system (Applied Biosystems). The temperature cycle started at 95 ^° C for 20 seconds, followed by 35 cycles of PCR stage at 95 ^° C for 1 second and 68 ^° C for 15 seconds, and post-PCR invader reaction for 99 ^° C for 30 seconds and 63 ^° C 2 Done in minutes. In the UGT1A1 * 28 single assay, it was necessary to change the annealing / extension temperature in PCR from 68 ° C to 64 ° C and the invader reaction temperature from 63 ° C to 60 ° C. This was thought to be due to the large difference between the calculated Tm value and the actual Tm value of the oligonucleotide, which affected the reaction. After completion of the reaction (25 minutes), SNP genotyping was performed by the auto-genotype-calling function of SDS 1.3.1 software (Applied Biosystems). Assays in which only a single homozygous cluster was detected in the Allele Discrimination (AD) plot could not be automatically clustered with this software, so these assays were manually genotyped. At this time, as a standard for manual call, normalized reporter signal value obtained by subtracting fluorescence signal value of negative control in AD plot {(FAM / Rox or Yakima Yellow / Rox in sample) − (in templateless control (NTC) FAM / Rox or Yakima Yellow / Rox)} adopted> 0.5.

5). Check genotype results in a sequence
To confirm the genotype results obtained from the 39 universal invader assays, we used ABI prism 3700 DNA Sequencer (Applied Biosystems) to sequence using purified genomic DNA samples from 18 volunteers. . BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems) was used for sequencing reactions according to the manufacturer's recommended protocol. Primers for PCR amplification and sequencing reactions are shown in Table 1 and SEQ ID NOs: 1-283.

6). Measurement of genomic DNA yield by alkaline lysis method To determine the yield of genomic DNA by the reported alkaline lysis method and the modified alkaline lysis method of the present invention, the QuantiBlot Human DNA Quantitation Kit (Applied Biosystems) Slot blot analysis was performed according to the manufacturer's recommended protocol.

Results Development and optimization of reaction solutions for the universal invader method In developing a reaction system using blood samples, it is necessary to investigate in detail the effects of various blood components on the reaction, and the composition of the reaction solution must be fully understood. It is important that Since the composition of a commercially available reaction solution for universal invader assay is not clear, the reaction solution was first developed. The previously reported alkali dissolution method uses Tris-HCl for neutralization after alkali dissolution [Rudbeck et al., Biotechniques 25: 588-592 (1998); Klintschar et al., Forensic Sci, 45: 669-673 (2000)], the pH value of the sample after neutralization is about 8.5. Furthermore, pH 8.5 was within the optimal range for both PCR and invader methods [Blanchard et al, PCR Methods Appl 2: 234-240 (1993); Kaiser et al, J Biol Chem 274: 21387 -21394 (1999)], 20 mM Tris-HCl pH 8.5 was employed as a reaction buffer for the universal invader assay.

The purified genomic DNA samples from 18 volunteers were then used to examine the effect of two important components Mg ²⁺ and monovalent cation concentration (Na ⁺ ) in the universal invader assay. Mg ²⁺ is an essential cofactor for the activity of Taq polymerase and Cleavase VIII. Further, Mg ²⁺ is essential for DNA polymerization and is known to form a complex with deoxynucleotide triphosphate (dNTP). It has also been reported that it has a strong effect on entropy and has an effect of increasing the Tm value of the oligonucleotide [von Ahsen et al., Clin Chem 47: 1956-1961 (2001)]. Increasing PCR primer Tm stabilizes PCR annealing and leads to efficient amplification of specific PCR products at appropriate concentrations (usually 1.5-10 mM), but nonspecific PCR products at higher concentrations It is known that the amplification of specific PCR products is inhibited. Monovalent cations have also been reported to influence entropy as strongly as Mg ²⁺ [von Ahsen et al., Clin Chem 47: 1956-1961 (2001)]. When the universal invader method was performed at various MgCl ₂ concentrations, neither PCR fragments nor fluorescent signals were observed at MgCl ₂ below 4.5 mM, and at concentrations higher than 10.5 mM, although PCR amplification was improved, a decrease in fluorescent signal was observed. It was. The optimum concentration range of MgCl ₂ that satisfactorily satisfies the PCR product and the fluorescence signal was 6.0-7.5 mM, respectively (FIG. 2). When MgCl ₂ was fixed at 7.5 mM and NaCl concentration was examined, sufficient PCR products were observed with NaCl exceeding 20 mM, but no fluorescence signal was observed at all. The optimal concentration range of NaCl sufficient to satisfy the PCR product and the fluorescent signal was 0-20 mM (FIG. 2). When 39 universal invader assays were performed under conditions of 7.5 mM MgCl ₂ and 0 mM NaCl, genotyping results at 100% call rate could be obtained (FIG. 3). All genotyping results were in complete agreement with the sequencing results. When 18 volunteers were used, some assays observed only a single homozygous cluster in the AD plot (CYP2C19 * 17, ABCC2-34T> C, CYP2C9 * 2, TPMT * 3A / B , TPMT * 3A / C, TPMT * 2, CYP2D6 * 4, CYP2D6 * 6, CYP2D6 * 18, CYP2D6 * 21, CYP2D6 * 44) for these assays, using HapMap samples or artificial templates for both alleles On the other hand, it was confirmed that each allele probe functions. Further evaluation was carried out by fixing MgCl ₂ to 7.5 mM and NaCl to 0 mM.

Universal Invader Method Combined with Alkaline Dissolution Method Reported Next, the universal invader method was evaluated using whole blood samples treated with the alkaline dissolution method reported above. 2 μl of treated whole blood sample and 8 μl of Universal Invader reaction solution were mixed and 39 assays were performed with a 25 min reaction time protocol. Of these, 25 assays gave genotyping results at 100% call rate, but 13 assays failed to determine genotypes due to PCR amplification errors or dot distribution variations in AD plots. Observed (FIG. 4). The total call rate was 86.32% (Table 2). These assay failures were predicted to be due to low yields of genomic DNA in the previously reported alkaline lysis method, and genomic DNA in the treated samples was quantified by slot blot analysis. As a result, the average value of 18 samples was 0.77 ng / μl (1.54 ng / well), and the lowest value was 0.32 ng / μl (0.64 ng / well). In the universal invader assay, genotype was possible with 0.5 ng / well of purified genomic DNA (FIG. 6), and the yield of genomic DNA was not considered to be the cause of direct assay failure. The present inventors speculated that certain inhibitory components in whole blood are still active to some extent even after alkaline treatment, and reducing PCR amplification and invader reaction may be the cause of direct failure (Fig. 5). Attempts were made to modify the previously reported alkaline lysis method to improve the performance of failed assays.

Modification of the previously reported alkali dissolution method (the present invention)
In order to remove the inhibitory component in the whole blood sample alkali-treated in a simple process, a method of precipitating the component with a concentrated Tris-HCl buffer and performing a simple centrifugation operation was attempted. High concentrations of Tris-HCl buffer have been reported to have the effect of precipitating proteins in the preparation of plasmid DNA from alkaline lysed bacterial cells [Chowdhury and Akaike, J Biotechnol 119: 343-347 (2006 )]. The present inventors considered that the same effect can be expected in blood components, and studied a method of neutralizing alkali-treated blood with a high concentration Tris-HCl buffer. First, various concentrations of Tris-HCl buffer (40, 80, 160, 240, 320, 400, 480, 560 mM Tris-HCl, pH 7.5) were prepared, and the concentration ratio of NaOH and Tris-HCl was kept constant. In this way, each concentration of Tris-HCl and alkali-treated blood was mixed in 180, 90, 45, 30, 22.5, 18, 15, and 13 μl of liquid volume (the liquid volume was determined in inverse proportion to the concentration). At this time, the pH value of each neutralization condition was almost constant (pH 8.5 to 8.7, room temperature). After simple centrifugation, a large amount of precipitate was observed when more than 80 mM Tris buffer was used for neutralization. As the precipitate was formed, an improvement in reactivity was observed in the assay that failed in the previously reported alkaline dissolution method (FIG. 6). The reactivity between samples in the universal invader assay varies slightly with the use of 80 mM Tris-HCl, and the fluorescence signal derived from the invader reaction tends to tilt downward when using more than 320 mM (FIG. 6). The optimal concentration range of Tris-HCl used in the experiment was 160 to 240 mM. For further evaluation, 160 mM Tris-HCl was used. Further, when the yield of genomic DNA in the protocol of the present invention was examined, the average of 18 samples was 1.14 ng / μl (2.28 ng / well), and the lowest sample was 0.5 ng / μl (1 ng / well). Met. These values were higher than the previously reported methods (Non-Patent Documents 14 and 15) and were sufficient values for the universal invader method. Using 19 whole blood samples treated with the alkaline lysis method of the present invention, 39 assays were performed with a reaction time of 25 minutes, and genotyping results were obtained at 100% call rate in all assays. (Figure 7, Table 2). There was no discrepancy in results between duplicate wells, and each genotype was in complete agreement with the results in the universal invader assay or sequencing using purified genomic DNA.

DISCUSSION The present inventors have invented a method capable of completing SNP genotyping within 30 minutes after blood collection by developing a reaction solution for universal invader assay and improving the neutralization method of alkali-treated blood. The genotyping result obtained by the method developed by the present inventors is completely consistent with the result of sequencing using purified DNA, and its accuracy has been proved. In addition, the protocol is simple, rapid, and easy to use, and it was considered advantageous for the development of microfluidics and automation equipment targeting POCT.

In this example, the present inventors first developed a reaction solution and found that the optimum range of Mg ²⁺ in the universal invader method was 6.0 to 7.5 mM. The reason is that in the universal invader reaction of the present invention, 1.6 mM dNTPs require the same concentration of free Mg ²⁺ and Taq DNA polymerase and Cleavase VIII are 1 mM and 4 mM for efficient enzyme activity, respectively. The above free Mg ²⁺ is required. As a result, the optimum range of Mg ²⁺ was 6.0 to 7.5 mM, and when it was less than 4.5 mM, it was considered that the PCR product and the fluorescence signal were not observed due to the lack of Mg ²⁺ . In Mg ²⁺ exceeding 10.5 mM, sufficient amplification of the PCR product was observed, but inhibition of the invader reaction was observed (FIG. 4). This result was thought to be due to the high concentration of Mg ²⁺ stabilizing the allele probe hybridization and reducing the efficiency of the cyclic cleavage reaction in the invader reaction. Therefore, it is very important to maintain the optimum concentration of Mg ²⁺ in the universal invader method. A negligible concentration of Mg ²⁺ (0.057 mM) may be introduced into the reaction solution from whole blood samples treated with the modified alkaline lysis protocol of the present invention [Blanchard et al, PCR Methods Appl 2: 234 -240 (1993)], the universal invader method was considered to have little effect.

In the study of NaCl concentration, when Mg ²⁺ concentration was fixed at 7.5 mM, we observed that the lower the NaCl concentration, the better the invader reaction, but with NaCl exceeding 20 mM, PCR amplification was improved. The invader reaction was observed to decrease (FIG. 5). As in the case of Mg ²⁺ , this result was thought to have been caused by stabilized hybridization of the allele probe with high concentrations of Na ⁺ . Although the reaction solution of the universal invader method does not contain monovalent cations, the alkaline solution (200 mM NaOH) and whole blood (124 mM Na ⁺ and K ⁺ ) contain a considerable amount of monovalent cations. Therefore, it was necessary to consider the introduction of monovalent cations from whole blood samples in protocol development. Under modified alkaline dissolution conditions, the concentration of monovalent cations is estimated to be about 13 mM as the final concentration and is strictly adjusted to 20 mM or less.

  As a method of omitting the genomic DNA purification step, the present inventors selected the alkaline lysis method. In recent years, it has been reported that the formamide extraction method is useful for TaqMan method and melting curve analysis. However, the heat treatment during extraction, the instability of formamide and the necessity of cryopreservation, the influence on the Tm value of oligonucleotides There are significant barriers to the realization of POCT, such as changes in reaction conditions. Compared to this, the alkali melting method, which can be processed at room temperature and does not require changing reaction conditions, was thought to be very useful in realizing POCT. In the alkaline dissolution method of the previous report (Non-Patent Documents 14 and 15), an inhibitory effect on the PCR reaction was recognized and was insufficient for practical use. However, the reactivity was improved by an improved method using high concentration Tris-HCl, A blood pretreatment method that can withstand practical use has been established. This improvement adds a step of removing precipitates, but it can be achieved by simple centrifugation and also possible by filtration, so it is not considered to be a significant barrier to realizing POCT. It is done.

  As a SNP genotyping technique from whole blood, the universal invader method has several advantages over melting curve analysis and TaqMan method. The universal invader method has a high target-specific recognition ability and is considered to have a higher S / N ratio than the above method based only on hybridization. Furthermore, since a high-cost fluorescent probe can be commonly used for any target SNP, it is more cost-effective than the above method that requires a fluorescent probe for each target. In addition, the universal invader method can be completed within 25 minutes, with the melting curve method (40 minutes) on the Light cycler (Roche Molecular Science) and the TaqMan method (2 hours on the ABI Prism 7700 (Applied Biosystems)). ) Is faster than. The format and simplicity of the single tube are equivalent to the above assay, and can be applied to POCT. With regard to assay design flexibility, as shown in this example, UGT1A1 * 28, one of the most important and difficult to establish assays in the field of pharmacogenomics, and other important polymorphisms are covered extensively. It can be said that it is enough. The universal invader method is considered to be one of the most promising SNP genotyping methods in POCT.

  In addition to melting curve analysis and TaqMan method, a method using "asymmetric isothermal amplification and a novel mismatch suppression technique" has been reported as one of the new rapid SNP genotyping methods from whole blood samples using alkaline lysis method [Mitani et al., Nat. Methods 4: 257-262 (2007)]. This technique has been reported to have a high S / N ratio and can be genotyped in 20-60 minutes from a whole blood sample. However, the principles of the art are very complex, and it may be necessary to further evaluate assay performance and assay design flexibility. With respect to many other reported techniques, to the best of our knowledge, there are few reports of SNP genotyping from whole blood samples.

  According to the present invention, a genotyping result can be obtained from a blood sample within 30 minutes accurately, simply and inexpensively. The method of the present invention is a promising method for the realization of individual medical care.

FIG. 1 shows the principle of SNP genotyping by the universal invader method. FIG. 2 is a diagram in which optimum conditions for MgCl ₂ concentration and NaCl concentration in the universal invader method were examined (eg, rs7294 (VKORC1)). (a) is a diagram of examining the optimal conditions for the MgCl ₂ concentration in the universal invader method. The upper row is an electrophoretic image of sample 1. The numbers in the figure shows the final concentration of MgCl _2. The arrow indicates the target amplicon (106 base). The lower row is an AD plot when 10 genomic DNA samples are used. (B) It is the figure which investigated the optimal conditions of NaCl concentration in a universal invader method (for example, rs7294 (VKORC1)). The upper row is an electrophoretic image of sample 1. The numbers in the figure shows the final concentration of MgCl _2. The arrow indicates the target amplicon (106 base). The lower row is an AD plot when 10 genomic DNA samples are used. FIG. 3 is an AD plot of 39 universal invader assays using 18 purified genomic DNA samples. All samples were reacted in duplicate. Alphanumeric characters in each AD plot indicate gene or allele name and polymorphism. All samples were reacted in duplicate. FAM and Yakima Yellow fluorescence signals were normalized with the Rox signal. The Y axis of each AD plot shows the normalized fluorescence intensity of the FAM allele. The X-axis shows the normalized fluorescence intensity of the Yakima Yellow allele. ■, ◆, black triangle, and ● indicate a control without template, a homozygote of FAM allele, a heterozygote of FAM allele and Yakima Yellow allele, and a homozygote of Yakima Yellow, respectively. FIG. 4 is an AD plot of 39 universal invader assays using 18 whole blood samples treated by the alkaline lysis method reported previously (comparative example). All samples were reacted in duplicate. Alphanumeric characters in each AD plot indicate gene or allele name and polymorphism. All samples were reacted in duplicate. FAM and Yakima Yellow fluorescence signals were normalized with the Rox signal. The Y axis of each AD plot shows the normalized fluorescence intensity of the FAM allele. The X-axis shows the normalized fluorescence intensity of the Yakima Yellow allele. ■, ◆, black triangle, and ● indicate a control without template, a homozygote of FAM allele, a heterozygote of FAM allele and Yakima Yellow allele, and a homozygote of Yakima Yellow, respectively. X indicates a specimen whose genotype could not be determined by automatic call. FIG. 5 shows an AD plot and an electrophoretic image of a universal invader assay (CYP2C9 * 3) using 18 whole blood samples treated by the alkaline lysis method described in the previous report (comparative example). FIG. 6 shows a modification of the previously reported alkali dissolution method. a: Precipitate formation by using high concentration of Tris-HCl to neutralize alkali-treated blood. The numerical values in the figure indicate the concentration of the neutralization buffer used, and 40 mM is the concentration of the previously reported neutralization buffer (40 mM Tris-HCl pH 7.5). In order to precipitate and remove the inhibitory whole blood components, various concentrations of neutralization buffer (Tris-HCl pH 7.5 in the range of 40-560 mM) can be used with the same concentration ratio of NaOH and Tris-HCl. The liquid volume was adjusted so as to be (in the range of 12.8 to 180 μl) and added to the alkali-treated blood solution. b: Effect of various neutralization conditions on the PCR reaction (electrophoresis images of sample 1 and sample 2 after universal invader reaction (CYP2C9 * 3)). c: Effect of various neutralization conditions on fluorescence amplification of universal invader assay (CYP2C9 * 3) (AD plot and real-time fluorescence data). Ten whole blood samples were used for evaluation. FAM and Yakima Yellow fluorescence signals were normalized with Rox signal. The Y-axis of each AD plot shows the normalized fluorescence intensity of the FAM allele. The X axis shows the normalized fluorescence intensity of the Yakima Yellow allele. ■, ◆, and black triangles indicate the templateless control, the FAM allele homozygote, and the FAM allele and Yakima Yellow allele heterozygote, respectively. X indicates a sample for which genotype could not be determined by automatic call. FIG. 7 is an AD plot when 39 universal invader assays were performed with a 25 minute reaction time protocol using 18 whole blood samples treated with the modified alkaline lysis method. All samples were reacted in duplicate. FAM and Yakima Yellow fluorescence signals were normalized with the Rox signal. The Y axis of each AD plot shows the normalized fluorescence intensity of the FAM allele. The X-axis shows the normalized fluorescence intensity of the Yakima Yellow allele. ■, ◆, black triangle, and ● indicate a control without template, a homozygote of FAM allele, a heterozygote of FAM allele and Yakima Yellow allele, and a homozygote of Yakima Yellow, respectively.

Claims (9)

A method for preparing a single nucleotide polymorphism genotyping test sample including the following steps:
Mixing 4 to 40 parts by volume of an aqueous solution of 100 to 200 mM alkali metal hydroxide with 1 part by volume of an animal-derived blood sample;
Adding 0.5 to 5 parts by volume of 80 to 560 mM Tris buffer to 1 part by volume of the mixed solution, and neutralizing;
And centrifuge or filter the neutralized solution to obtain a test sample. Testing method for single nucleotide polymorphism genotyping including the following steps:
Mixing 4 to 40 parts by volume of an aqueous solution of 100 to 200 mM alkali metal hydroxide with 1 part by volume of an animal-derived blood sample;
Adding 0.5 to 5 parts by volume of 80 to 560 mM Tris buffer to 1 part by volume of the mixed solution, and neutralizing;
Centrifuge or filter the neutralized solution to obtain a test sample, and genotyping by the universal invader method using the test sample.   The process according to claim 1 or 2, wherein the alkali metal hydroxide is sodium hydroxide or potassium hydroxide.   The method according to claim 1 or 2, wherein the Tris buffer is Tris-HCl (pH 7 to 9, 25 ° C).   The method according to claim 1, wherein the process from the mixing step to the step of obtaining a test sample is performed within 5 minutes. The method according to claim _2, wherein the universal invader method is performed in the presence of 4.5 to 9.0 mM MgCl ₂ and 0 to 20 mM NaCl.   The method according to any one of claims 2 to 6, wherein the mixing step to the genotyping step are performed within 30 minutes. It comprises an aqueous solution of alkali metal hydroxide and 80～560MM Tris buffer of 100 to 200 mm, single nucleotide from blood specimens from animals polymorphism kit for preparing a genotyping inspection sample. The kit according to claim 8, further comprising a mixing container and / or a centrifuge tube.