JP2006061041A - Method for extracting nucleic acid and kit for extracting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for extracting a nucleic acid without using reagents which require a careful handling, independently from kinds of samples and hardly affecting following analyses. <P>SOLUTION: This invention comprises the method for extracting the nucleic acid comprising a process contacting samples to be tested with glycocholic acid. The invention also relates to the above method for extracting the nucleic acid, wherein the concentration of the glycocholic acid is 1-10 mM. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nucleic acid extraction method.

In the conventional nucleic acid extraction method, many steps such as removing the cell wall and cell membrane after removing them, denaturing the protein, and separating the nucleic acid are required. Reagents used at this time include those requiring careful handling such as strongly alkaline sodium hydroxide solution and highly corrosive phenol solution.
In order to solve this problem, a method for purifying DNA from a mixture of microbial cells and phages has been proposed (see, for example, Patent Document 1). Specifically, in this method, instead of the DNA concentration step using phenol and ethanol, a step of passing the sample through an ultrafiltration membrane is provided to remove impurities and concentrate the DNA.
JP-A-5-236963

  However, according to the conventional DNA extraction method described above, a strong alkaline sodium hydroxide solution is necessary for purification even though the use of a phenol solution can be avoided. As described above, there is a problem that even though the types of reagents that require careful handling can be reduced, it is difficult to replace all the reagents with those that are easy to handle. Furthermore, it is relatively easy to purify DNA from a mixture of microbial cells, specifically Escherichia coli, and phage, since there are few contaminants and there are few obstacles such as cell walls. In the case of application, it is difficult to provide a method for efficiently extracting nucleic acids regardless of the type of living organisms, because the impurities contained in the organism-specific structures and samples become obstacles.

  In addition, reagents remaining in nucleic acid samples extracted using conventional extraction methods may interfere with subsequent analysis, requiring additional purification steps and reducing analysis accuracy. It was. In particular, at present, in the field of biotechnology, it is recognized that a method for efficiently and intactly extracting genetic DNA from cells is essential for genetic testing of organisms. Also, in order to expedite research, automation of gene extraction and other operations has been attempted. However, as the number of processes increases, the steps involved in purification such as centrifugation and suction filtration become complicated, and automatic genetic testing equipment It was an obstacle to labor saving and miniaturization.

  Therefore, in view of the above problems, the object of the present invention is to perform processing in a short time without using a reagent having careful handling, regardless of the type of test sample, and to affect the subsequent analysis. It is an object of the present invention to provide a nucleic acid extraction method that is difficult to give.

  The characteristic means of the nucleic acid extraction method of the present invention for achieving this object is that, as described in claim 1, it comprises a step of bringing glycocholic acid into contact with the test sample.

In the above characteristic means, as described in claim 2, the concentration of the glycocholic acid is preferably 1 to 10 mM,
As described in claim 3, it is preferable to have a step of contacting a proteolytic enzyme with the test sample,
As described in claim 4, the test sample is preferably an animal-derived sample,
As described in claim 5, it is preferable that the test sample is a sample containing microbial cells,
As described in claim 6, it is preferable that the microorganism is Cryptosporidium.

  In order to achieve this object, the nucleic acid extraction kit according to the present invention is characterized in that it contains glycocholic acid as described in claim 7.

In the above-mentioned characteristic configuration, as described in claim 8, it is preferable that glycocholic acid is contained so that the final concentration is 1 to 10 mM at the time of use,
As described in claim 9, it is preferable to include a proteolytic enzyme.

  The nucleic acid extraction method according to the present invention focuses on bile acids, which are one of digestive fluids possessed by mammals. The main components of bile acids are cholic acids such as taurocholic acid, deoxycholic acid and glycocholic acid. Cholic acids also have properties as surfactants, and in particular, deoxycholic acid is sometimes used as a cell membrane disrupting reagent for the extraction of membrane proteins. However, taurocholic acid and deoxycholic acid known so far strongly inhibit enzyme reactions such as PCR, and thus must be removed after cell destruction reaction. The inventors have found that glycocholic acid hardly inhibits the gene amplification reaction even when the same cholic acid is used.

The molecular chemical mechanism of cell destruction of glycocholic acid is presumed as follows. The cell membrane is composed of phospholipids and proteins, and glycocholic acid enters the lipid bilayer of the phospholipids of the cell membrane and makes the membrane fragile. This mechanism will be common to all living organisms regardless of whether they are animals, plants or microorganisms. Therefore, this method is considered to exert its effect on test samples derived from a wide range of organisms.
Furthermore, glycocholic acid is easier to handle than phenolic solutions and strong alkaline solutions. At the same time, since the enzyme reaction after DNA extraction is not hindered, the sample subjected to the reaction does not require a purification step. As a result, the nucleic acid extraction process can be performed in a short time, and labor saving and downsizing of the automatic genetic testing device are realized.

Therefore, as described in claim 1, the nucleic acid extraction method having the step of bringing glycocholic acid into contact with the test sample performs the treatment in a short time without using a reagent having careful handling. Therefore, it is considered to be very useful for automation and labor saving of DNA extraction reaction.
In the present application, “nucleic acid” is used as a concept including DNA, RNA, and derivatives thereof.

  Furthermore, as described in claim 2, when the concentration of the glycocholic acid at the time of contacting with the test sample is 1 to 10 mM, the enzyme reaction after the extraction is surely inhibited without inhibiting the nucleic acid. Can be extracted and is most efficient.

  As described in claim 3, when the proteolytic enzyme is brought into contact with the test sample, the protein mixed in the sample is decomposed, so that the molecular structure of the membrane is completely destroyed and more reliably. Can destroy cells. Furthermore, it is often heated when a proteolytic enzyme is allowed to act, and it is considered that the destruction of the cell membrane proceeds due to fluctuation caused by heat simultaneously with the decomposition of the protein.

  As described in claim 4, since an animal-derived sample can be used as a test sample, for example, it can be used in a medical field such as disease diagnosis, and a rapid and highly accurate diagnostic method. Can be provided.

  Samples containing microbial cells may be difficult to extract rapidly and efficiently due to obstacles such as a robust structure peculiar to the organism or a multi-layered membrane structure. As described above, the present method can be applied even to these microorganisms. Therefore, by adopting this method, it is possible to achieve high speed and high accuracy even in the medical field such as diagnosing diseases and inspecting for environmental pollution.

As described in claim 6, if the microorganism contained in the test sample is Cryptosporidium, it can be used for safety inspection of tap water and the like, which is very useful from an epidemiological viewpoint. The Cryptosporidium is a parasitic protozoan parasitic on the digestive tract of mammals such as humans, cattle and pigs, and mammals such as dogs and mice belonging to the order of the spore genus Coccidium. When humans are infected, symptoms such as watery or mucous stool accompanied by abdominal pain persist for about 3 days to 1 week, sometimes with vomiting and fever. Since the enormous number of oocysts excreted together with feces from infected patients and infected animals is a source of infection to new individuals, the transmission route of Cryptosporidium is diverse. Cryptosporidium oocysts present in the water are resistant to chlorine and chlorination in water treatment is ineffective, so it is difficult to remove them completely by normal water treatment. Infection may occur.
Various methods have been proposed to determine the type and amount of Cryptosporidium, and there was also a method of extracting and identifying and quantifying nucleic acids from oocysts by PCR. In this method, by freezing and thawing, sonication, etc., after removing the sporozoite from the oocyst, by dissolving the sporozoite with a protease, or by known DNA purification treatment such as phenol extraction / ethanol precipitation, It was common to do. For this reason, various processes are complicated and time-consuming, and in the process of DNA purification, there is always a problem of loss of DNA, leading to a decrease in detection sensitivity.

  However, observing the decapsulation process by this method under a microscope, it was confirmed that by contacting glycocholic acid with Cryptosporidium oocysts, not only the oocysts but also the sporozoites that came out of them were destroyed. It was. Therefore, in the present method, when the test sample is Cryptosporidium, the operation is simplified and rapid analysis is possible in the test, and the detection accuracy is improved.

  In carrying out the nucleic acid extraction method, as described in claim 7, when a nucleic acid extraction kit containing glycocholic acid is used, the treatment can be performed in a short time without using a reagent having careful handling. It can greatly help automate the DNA extraction reaction and save labor.

  Furthermore, as described in claim 8, the nucleic acid extraction kit includes glycocholic acid so that the final concentration is 1 to 10 mM at the time of use, thereby reliably inhibiting the enzymatic reaction after extraction. Without extraction, the nucleic acid can be extracted.

  Furthermore, as described in claim 9, when the nucleic acid extraction kit contains a proteolytic enzyme, the protein mixed in the sample is degraded, so that the cells can be more reliably destroyed.

Embodiments of the present invention will be described below.
The nucleic acid extraction method according to the present invention includes a step of bringing glycocholic acid into contact with a test sample, and preferably, contacting the glycocholic acid with the test sample in a state where the concentration is 1 to 10 mM. Let This process is continued until the cell membrane of the test sample is sufficiently destroyed. For preparing a small amount of sample such as a PCR sample, a contact time of about 5 to 60 minutes is sufficient.

  In order to ensure destruction of the cell membrane of the test sample, a step of bringing a proteolytic enzyme into contact with the test sample can be further provided. This step can be performed simultaneously with the step of bringing glycocholic acid into contact with the test sample. For example, proteinase K is suitable as the proteolytic enzyme, and for example, 50 to 1200 mAU / ml is added.

  This method can be applied to all living organisms regardless of whether they are animals (including humans), plants, or microorganisms (including protozoa and protozoa). Specifically, if a nucleic acid is extracted from a test sample derived from an animal such as blood containing blood cells and a biopsy sample containing some animal cells, it can be used for examinations in the medical field. If nucleic acid is extracted from a plant-derived test sample, it can be used to identify varieties and brands. A test sample containing microorganisms can be used for examining environmental pollution by microorganisms, or in the field of bioremediation, for confirming the presence of pollutant assimilating bacteria in the soil to be purified. Would be available to you. In addition, these uses are illustrations, Comprising: It is not limited to the above-mentioned thing.

  Here, when nucleic acid is extracted from organisms having cell walls such as plant cells and yeasts such as molds and Gram-positive bacteria, a pretreatment step for decomposing the cell walls is provided before contacting the test sample with glycocholic acid. It is preferable. Since the composition of the cell wall differs depending on the species, a cell wall degrading enzyme (for example, cellulase, zymolyase, lysozyme, etc.) corresponding to each composition is brought into contact with the test sample to lyse the cell wall. However, this method can be applied without cell wall degrading enzymes acting on these organisms.

  When carrying out the nucleic acid extraction method according to the present invention, each step may be executed by a mechanical device and automated. In such a case or when a person performs an operation, a reagent containing glycocholic acid can be supplied as a nucleic acid extraction kit for the convenience of the person. In this kit, it is preferable to blend glycocholic acid so that the final concentration during use is 1 to 10 mM. Furthermore, a cell wall degrading enzyme and / or a proteolytic enzyme can be included in this nucleic acid extraction kit. For example, they can be supplied as a stock or concentrated solution of a liquid (solution) dissolved and sealed together with a reagent such as a buffer required for each step.

  As an analysis example using the nucleic acid extraction method according to the present invention, a method for detecting species of Cryptosporidium will be described below. In this method, in order to prepare a nucleic acid sample from a test sample, a step of using the nucleic acid extraction method according to the present invention, and a single nucleotide variation between species of a species belonging to the genus Cryptosporidium is specified in the prepared nucleic acid sample. A step of hybridizing with the designed probe, a step of detecting the hybridization between the DNA and each probe using a label as an index, and a step of analyzing the detection result. Furthermore, in the process of preparing a nucleic acid sample from a test sample, a glycocholic acid treatment is performed, followed by a protease treatment, thereby improving nucleic acid extraction efficiency and shortening the time required for extraction. And achieve rapid detection. Here, species-identifying detection means detecting Cryptosporidium as well as Cryptosporidium species, and species discrimination is substantially different from other species belonging to the genus Cryptosporidium. It is meant to identify a group belonging to a certain species.

  Here, Cryptosporidium to be detected refers to a microorganism belonging to the genus Cryptosporidium, and specifically Cryptosporidium parvum species (hereinafter abbreviated as C. parvum), Cryptosporidium. muris species (hereinafter abbreviated as C. muris), Cryptosporidium baileyi species (hereinafter abbreviated as C. baileyi), Cryptosporidium serpentis species (hereinafter abbreviated as C. serpentis), Cryptosporidium meleagridis species (hereinafter abbreviated as “C. baileyi”) C. meleagridis)), Cryptosporidium felis species (hereinafter abbreviated as C. feris), etc., and the method of the present invention is used for preparing nucleic acids for species-specific detection of these species. Used for.

  The test sample used here is a concept including any sample suspected of being contaminated with Cryptosporidium present in the environment or in the living body. Water samples existing in the environment such as raw water, river water, lake water, well water, ground water, sewage, waste water, pool water, park water, or environment such as ranch soil, farmland soil, lake soil, river soil Examples include environmental samples such as soil samples, biological samples such as stool such as humans, livestock, and pets, food samples such as beverage foods, raw vegetables, and fruits, but are not limited thereto is not.

  The step of preparing the nucleic acid sample from the test sample is performed by recovering the oocysts of Cryptosporidium from the test sample and extracting the chromosomal DNA from the recovered oocysts. Recovery of oocysts from the test sample can be performed using any known method. In the case of a stool sample, for example, a method in which stool is extracted with diethyl ether and then purified by sucrose density gradient centrifugation is exemplified. In the case of a water sample, for example, 20 l of river water is passed through a metal mesh and is 1 cm or more. After removing the substance, use a cellulose mixed ester membrane filter (pore size 1.2 μm: manufactured by Millipore) with a diameter of 90 or 142 mm to make 500 μl to 2 ml, and take 10 to 50 μl of it directly. Can be used in the method of the present invention.

  Extraction of chromosomal DNA from oocysts is preferably performed with glycocholic acid and then with protease. In particular, it is preferable to treat with glycocholic acid and then with proteinase K. The concentration of the reagent used in the present invention is preferably 1 to 10 mM when glycocholic acid is used. The condition of the glycocholic acid treatment is preferably 5 to 240 minutes at 24 to 40 ° C., particularly preferably 15 minutes at 37 ° C. Moreover, the proteinase K treatment after deoxycholic acid treatment is preferably performed in the presence of 1 to 50 mM EDTA, preferably 10 mM, and the treatment conditions are 5 to 37 ° C. 60 minutes is preferable, and particularly preferably, incubation is performed at 70 ° C. for 10 minutes, followed by further incubation at 94 ° C. for 10 minutes. In addition, since glycocholic acid is not a substance that inhibits the PCR reaction like taurocholic acid, the sample treated by the method of the present invention can be directly amplified without going through the purification process of the extracted DNA. Compared with the treatment with a mixed solution of taurocholic acid and trypsin that has been reported in the past (for example, see JP-A-2001-252099), each treatment time can be greatly reduced. In combination with this, the time required for the extraction process can be shortened. In addition, nucleic acid loss is inevitably caused in the course of DNA purification, and the recovery rate of DNA after purification is at most about 80%. However, according to the present invention, the loss of DNA due to DNA purification is reduced. Since it can be minimized, it is possible to maintain a DNA extraction efficiency of almost 100%, and it is possible to achieve a final improvement in detection efficiency.

  A nucleic acid sample extracted from oocysts is preferably amplified in order to improve detection sensitivity. Although amplification of DNA is generally performed by PCR, the present invention is not limited to this. Hereinafter, amplification using PCR will be described in detail.

PCR can be performed according to a known PCR method using the DNA obtained above as a template, and is preferably performed in two steps. The second PCR is an amplification obtained by the amplification reaction obtained in the first PCR. Preferably, the product is used as a template. In the second PCR, it is particularly preferable to incorporate a labeling reaction for the sequence to be amplified, and the label will be described in detail later. In addition, the primer used for PCR is designed based on the target base sequence (the expression of target sequence or target nucleic acid may also be used), in this case, the base sequence of Cryptosporidium. Yes, such base sequences are obtained by searching gene databases such as DNA Databank of Japan (DDBJ), GenBank, European Molecular Biology Laboratory (EMBL), in which the base sequences of Cryptosporidium genes are registered. be able to. Since PCR amplification efficiency and specificity depend on the primer setting position and primer sequence, it is a sequence that can efficiently amplify the base sequence of Cryptosporidium gene, and any species of Cryptosporidium Since amplification is preferable, it is preferable to design based on a highly conserved sequence common to Cryptosporidium species. If PCR is performed in two stages, the second PCR should be performed by nested PCR with primers set inside the target sequence amplified in the first PCR in order to eliminate non-specific amplification products. Can do. As primers used for this nested PCR, in the first PCR, 5′-GGAACCTGGTTGATCCTGCCAG-3 ′ as a sense primer; 5′-GGTCGATCCCCTAACTTTCGTT-3 ′ as an antisense primer; In the second PCR, 5′-TCAAAGATTAAGCCATGCATGTCT-3 ′; SEQ ID NO: 3 is preferred as the sense primer, and 5′-GATTAATGAAAACATCCTTGGCAAA-3 ′ sequence ID No. 4) is preferred as the antisense primer. However, it is not limited to these. The amplification product after PCR amplification is preferably purified by a known means such as gel filtration, then fragmented by DNase, sonication, Mg ²⁺ ion treatment, etc., and subjected to a hybridization experiment described later.

  The oligonucleotide primer can be chemically synthesized. For example, it can be chemically synthesized by solid phase synthesis using the known phosphoramidite method (Nucleic Acids Research, 17, 7059-7081 1989), and the desired nucleotide sequence can be obtained by a commercially available automatic nucleic acid synthesizer. It is also possible to automatically synthesize an oligonucleotide consisting of The synthesized oligonucleotide is purified by a known method such as HPLC as necessary.

  In order to detect Cryptosporidium species-specifically based on single-base mutations that occur between species, the probe set group uses each of the species belonging to the genus Cryptosporidium using known gene databases such as DDBJ, GenBank, and EMBL. The base sequence of the 18S ribosomal gene is searched, a species-specific single nucleotide mutation is identified from each homology analysis, and it is designed based on the identified single nucleotide mutation. The site where the mutation has occurred is used as a detection site for species-identifying detection, and the base of the site identified as showing a single base mutation has 4 different mutations (a, g, c, t) 4 Each type of probe was set as one set and included at least one set. Each probe constituting the probe set group designed in this way is brought into contact with the target nucleic acid, and the probe having the mutated base in one probe set consisting of four types of probes in which one base is mutated is most bound. By detecting whether or not, Cryptosporidium is detected species-specifically.

  Particularly preferably usable probe set groups are summarized in Table 1, but are not limited thereto. Moreover, it can comprise as what has a part probe set among these probe set groups, and can be suitably selected according to the detection objective etc. In these sequences, the base indicated in lower case is a base showing a single base mutation.

  In addition, the probes constituting the probe set group do not necessarily have to be designed with all of the sequences shown in Table 1 or their complementary sequences as probes, and select appropriate regions by selecting Tm values and the like. Use the length. However, since the probe discriminates a species based on a species-specific single nucleotide mutation, it is necessary to detect a single nucleotide mutation with specificity. Therefore, in the sequence shown in Table 1 or a complementary sequence thereof, It must have a length of at least 10 consecutive bases, more preferably 18 bases or more, including a base sequence consisting of a base exhibiting a single base mutation and 3 bases on the 5 ′ side and 3 bases on its 3 ′ side There is. Therefore, as long as these conditions are satisfied, a sequence having an appropriate length may be selected from the sequences in the sequence listing, and base sequences other than those listed in Table 1 can be added. In this case, it is necessary to select and add a sequence that does not allow hybridization other than the species-specific region. The length of the probe is preferably 10 to 70 bp, particularly preferably 10 to 40 bp, from the viewpoint of stability of the hybrid with the target nucleic acid and ease of handling. In addition, it is preferable that the base exhibiting a species-specific single nucleotide mutation is designed so as to be located in the center of the oligonucleotide constituting the probe. From such a viewpoint, the probe set group is designed.

  Here, the probe in this specification means a region that hybridizes with a target nucleic acid, and a chemical structure other than nucleotides may be further added to the nucleic acid molecule actually used as a probe. For example, when detection is performed by immobilizing a probe, for example, by producing a microarray immobilized on a probe solid phase carrier and performing detection, the functional group for the immobilization has the 3 ′ end of the probe or Examples include a form introduced into the hydroxyl portion at the 5 ′ end, and further to the base portion and the phosphodiester portion.

  The probe used here can be chemically synthesized. For example, it can be chemically synthesized by solid phase synthesis using the known phosphoramidite method (Nucleic Acids Research, 17, 7059-7081 1989), and the desired nucleotide sequence can be obtained by a commercially available automatic nucleic acid synthesizer. It is also possible to automatically synthesize an oligonucleotide consisting of The synthesized oligonucleotide is purified by a known method such as HPLC as necessary.

Either the target nucleic acid or the probe to be detected is labeled, and the method for labeling the nucleic acid is known, and is labeled by a method suitable for the type of label. As will be described later, it is preferable to label the target nucleic acid to be detected. As labels, fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, Cy3 and Cy5, radioisotopes such as ³⁵ P, ¹³¹ I and ³⁵ S, enzymes such as alkaline phosphatase and horseradish peroxidase, etc. Any label that can be used for can be used. In addition, labeling with an organic compound such as digoxigenin or biotin can also be preferably exemplified, and the enzyme binds to the nucleic acid via the antibody by immunologically binding with a digoxigenin antibody labeled with alkaline phosphatase or the like. The detection can be performed by adding a chromogenic substrate such as tetrazolium blue to detect the color, and in the case of biotin, the reaction can be performed in the same manner by utilizing the interaction with streptavidin.

  The labeling is preferably performed by introducing a label into the amplification product during the amplification reaction. As a method of introducing a label at the time of nucleic acid amplification, particularly preferably, an amplification product in which the inside is randomly labeled by performing an amplification reaction using a fluorescently labeled nucleotide such as Cy3-dCTP and incorporating the label into the amplification product. The method of creating can be used.

  The step of hybridizing the target nucleic acid prepared as described above with the probes constituting the probe set group can be performed according to all known hybridization techniques such as reverse dot blotting and dot blotting. Preferably, either the target nucleic acid or the probe is immobilized on a solid phase carrier using a known method. As the carrier used for fixation, any known carrier such as a microwell, a bead, a slide glass, or a membrane can be used, and a microarray is preferably exemplified. Although it is possible to immobilize the target nucleic acid to be detected and hybridize with the labeled probe, the method of contacting the hybridized target nucleic acid with the probe immobilized on the solid phase is easy to operate. From the viewpoint of When it is necessary to process a large number of specimens as in environmental diagnosis and clinical examination, a common immobilized probe can be used for each detection, and a kit, preferably a microarray, in which these probes are immobilized. It is particularly preferable in that it can be easily inspected by producing a large amount of.

  Hybridization conditions and washing conditions are appropriately selected and set empirically depending on the length of the sequence to be hybridized and the content of the GC nucleotide sequence. However, it is possible to appropriately select conditions that specifically hybridize and exhibit sufficient detection sensitivity and specificity. Well established formulas for variations of such hybridization conditions are well known in the art, for example, Sambrook et al, “Molecular Cloning: A Laboratory Manual”, 2nd edition, p. 9.47-9.51. , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory (1989). In particular, the present invention is a detection method for identifying species by detecting differences in hybridization intensity based on mutations at a single base level, and thus detecting such mutations at a single base level. It is necessary to set the conditions so that they fit. Examples of specific hybridization conditions and washing conditions include those described in the examples, but are not limited thereto.

  Detection of the nucleic acid hybridized to the probe is performed by hybridizing the probe of the present invention to a nucleic acid sample, removing unbound nucleic acid, and then detecting the nucleic acid hybridized to each probe using the label as an indicator. This can be detected by measuring radiation, color development, fluorescence, and luminescence using a known method corresponding to the type of label.

  The step of analyzing the detection result is performed by using four types of probes configured to have four different mutations (a, g, c, t) as one probe set in a base site showing a species-specific single nucleotide mutation. Since it constitutes, cryptosporidium-derived DNA is strongly bound to any one of the four types of probes, and this is performed. Here, the probe that binds most strongly in one probe set is defined as a sequence complementary to the target nucleic acid. The probe used for detection is set to have 4 mutation sites per single-base mutation of Cryptosporidium species-specific, and this is used as a detection site for species identification. The complementary sequence differs for each species, and Cryptosporidium species can be identified by detecting the complementary sequence. Therefore, the detection sensitivity and the detection accuracy improve as the number of detection parts increases. The probe set group exemplified in Table 1 as being particularly preferably usable is composed of 10 probe sets in which at least two detection sites are set for one kind of Cryptosporidium, which is particularly preferable. It is possible to detect species with high accuracy and high sensitivity. The detected label is preferably quantified, and the sum of the label intensities such as fluorescence intensities observed in each hybridization of the four types of probes configured as a probe set and the target nucleic acid is defined as 100%. It is preferable to evaluate by calculating the relative label intensity for each, and in the examples shown below, the evaluation is performed with a probe having a relative label intensity of 60% or more as a complementary sequence.

  In addition, by quantifying the amount of label to be observed, Cryptosporidium contained in the test sample can be quantified. Therefore, the detection in this specification includes quantitative detection.

  Each specimen of Cryptosporidium is hybridized with the probe in the same manner as the sample to be detected, and the observed hybridization pattern or complementary sequence is compared to detect and identify the species. Alternatively, it may be configured such that the hybridization pattern or complementary sequence information for each species is made into a database to automatically detect and identify the species.

  Detection is preferably performed by adding an appropriate positive control probe and negative control probe. The negative control probe needs to be an oligonucleotide sequence designed not to be complementary to any species of Cryptosporidium, for example, one designed based on the gene sequence unique to E. coli can be used, Specifically, 5′-AGAGTTTGATCCTGGCTCAG-3 ′ (sequence identification number 55) prepared from the sequence of the Escherichia coli 16S ribosomal gene can be exemplified. The positive control probe must also be an oligonucleotide sequence designed to be complementary to any species of Cryptosporidium, for example, based on consensus sequences conserved in all species of Cryptosporidium The designed one can be used, and specifically, 5′-GGAACCTGGTTGATCCTGCCAG-3 ′ (SEQ ID NO: 56) produced based on the consensus sequence of the 18S ribosomal gene of Cryptosporidium can be exemplified.

  Hereinafter, a method for carrying out the species-identifying detection method using Cryptosporidium nucleic acid extracted using the present invention using a microarray will be described in detail.

  In the microarray, the probe group of the present invention is appropriately arranged and fixed on a solid phase carrier. In particular, from the viewpoint of facilitating analysis, it is preferable that four types of probes configured to have four types of mutation sites per single base mutation constituting the probe set are arranged in a line. It is also possible to arrange appropriate positive probes and negative probes. As the microarray carrier, a glass plate, a quartz plate, a silicon wafer, and the like are preferably exemplified, but any known material conventionally used can be used.

  Any known method can be used to immobilize the probe on the carrier, and an optimum method is appropriately selected according to the type of probe and the type of carrier. Specific methods include electrostatic bonding to the carrier surface with polycations such as polylysine and polyethyleneimine using the charge of DNA, and surfaces with various silane coupling agents having amino groups, aldehyde groups, epoxy groups, etc. Preferred examples include a method of covalently bonding the treated carrier to DNA having an amino group, an aldehyde group, an SH group, biotin or the like introduced as a functional group at the terminal. Thereafter, treatments such as crosslink formation by ultraviolet irradiation, surface blocking, and washing are performed as necessary. In addition, the microarray can be produced by outsourcing to a contractor.

  The method for detecting Cryptosporidium using the microarray prepared above causes the nucleic acid sample prepared as described above to act on the microarray and hybridizes the probe immobilized on the carrier with the DNA contained in the nucleic acid sample. . The hybridization conditions have already been described above.

  After hybridization, DNA hybridized with the probe on the microarray is detected using a labeling substance as an index. When the fluorescent labeling is performed, the fluorescent image scanner is used. When the radioisotope labeling is performed, the fluorescent labeling is performed with an RI image scanner, and the amount of the labeling is measured by computer analysis. For example, fluorescence can be measured by detecting fluorescence with a fluorescence laser microscope and a CCD camera and analyzing the fluorescence intensity with a computer. , Evaluate.

  Reagents necessary for hybridization, reagents necessary for detection, and the like can be included in the kit in which each probe constituting the probe set group is immobilized, and is preferably in the form of a microarray.

  Examples of the present invention will be described below by taking DNA extraction from Cryptosporidium oocysts and a series of analyzes using the extracted DNA as examples. Unless otherwise specified, as deoxycholic acid, sodium deoxycholate monohydrate (special grade GR10712-12) manufactured by Nacalai Tesque was used. As glycoglycic acid, sodium glycocholate (360512 Cal 96% (TLC) 17135-01) manufactured by Nacalai Tesque was used. As the proteolytic enzyme, proteinase K manufactured by Nacalai Tesque was used. As a PCR reagent, Takara Bio TaKaRa Ex Taq (RR001A) was used.

[Examination Example 1] Examination of DNA extraction method from Cryptosporidium oocysts Glycocholic acid / deoxycholic acid treatment followed by protease treatment (hereinafter abbreviated as glycocholic acid / deoxycholic acid treatment / protease treatment) .) Comparison was made between DNA extraction efficiency when DNA was extracted from oocysts and DNA extraction efficiency when DNA was extracted based on the conventional DNA purification method.

  For glycocholic acid / deoxycholic acid treatment / protease treatment, deoxycholic acid is added to 10 μl of oocyst suspension PBS buffer of 2500 oocysts / μl to a final concentration of 0.1 mM or glycocholic acid to a final concentration of 1 mM. After incubation at 37 ° C. for 15 minutes, proteinase K (600 mAU / ml, manufactured by QIAGEN) and EDTA (final concentration 10 mM) were added, incubated at 70 ° C. for 10 minutes, and further incubated at 94 ° C. for 10 minutes. This was done by incubating for a minute. For DNA extraction based on the conventional DNA purification method, DNA was extracted from 50 μl of the above oocyst suspension PBS Buffer using QIAGEN genomic DNA kit (QIAGEN) according to the supplier's instructions.

  Next, 2.2 µl and 2 µl of each DNA sample extracted by glycocholic acid / deoxycholic acid treatment / protease treatment and conventional DNA purification methods are taken, and PCR reaction (25 µl total volume) is performed under the same conditions. FIG. 1 shows the result of visualizing the amount of DNA extracted by gel electrophoresis. Lane 1 is a band derived from a DNA sample prepared by a treatment method based on a conventional DNA purification method, and Lane 2 is derived from a DNA sample prepared by a treatment method based on glycocholic acid / deoxycholic acid treatment / protease treatment. Shows the band. From these results, it was found that glycocholic acid / deoxycholic acid treatment / protease treatment is a more effective means for extracting chromosomal DNA from oocysts because it has higher DNA extraction efficiency than conventional DNA purification methods. did.

  In addition, although not shown here, when observing the capsulation process in the above-mentioned glycocholic acid / deoxycholic acid treatment / protease treatment with a microscope, glycocholic acid not only destroys oocysts, but also sporozoites that emerge from it. It was confirmed that Therefore, it is presumed that the DNA extraction efficiency is increased because a completely fine cell structure is destroyed together with the effect of protease treatment.

[Examination Example 2] Examination of influence of taurocholic acid, glycocholic acid, and deoxycholic acid on PCR reaction By treating oocysts with each bile acid component of taurocholic acid, glycocholic acid, and deoxycholic acid, the oocysts of Cryptosporidium When DNA was extracted, the effect of these bile acid components on the amplification reaction by PCR was examined. The study was set up to amplify approximately 100 bases of different base sequences of the 18S ribosomal gene of Cryptosporidium in samples from which these bile acid components were removed by DNA purification and samples from which these components were not removed 2 Primer pair (18F 5'-TGTCACTACCTCCCTGTATTAGGATTG-3 '; SEQ ID NO: 57 and 95R 5'-CCTGAGAAACGGCTACCACATC-3'; SEQ ID NO: 58, 96F 5'-CTCCCTCTCCGGAATCGAA-3 '; SEQ ID NO: 59 and 174R 5'- CAGCTTTAGACGGTAGGGTATTGG-3 ′; SEQ ID NO: 60) was used for amplification reaction, and the amplification product was recognized by gel electrophoresis. 10 μl of 2500 oocyst / μl oocyst suspension was treated with 10 μl each of 70 mM glycocholic acid, deoxycholic acid and taurocholic acid at 37 ° C. for 30 minutes. 20 μl of 50% Triton X-100, 10 mM EDTA was added to this and reacted at room temperature for 5 minutes, 5 μl of which was subjected to PCR to obtain a PCR product of an unpurified sample. Another 20 μl was purified using a GFX DNA purification kit (Pharmacia) to obtain an equal amount of 20 μl of purified DNA solution. Of these, 5 μl was subjected to PCR to obtain a PCR product of a purified sample. The result is shown in FIG. 100 bp (18F-95R) is derived from the unpurified sample with the primer pair of SEQ ID NO: 57 and 58, and Purified (18F-95R) is derived from the purified sample with the primer pair of SEQ ID NO: 57 and 58. Amplification product, 100 bp (96F-174R) is an amplification product derived from an unpurified sample with a primer pair of SEQ ID NO: 59 and 60, and Purified (96F-174R) is a primer pair of SEQ ID NO: 59 and 60 An amplification product derived from the purified sample.

  As a result, in the case of treatment with deoxycholic acid or glycocholic acid in any amplification, the amount of amplified product derived from the sample that did not undergo the DNA purification process was larger, whereas taurocholic acid treatment In the case of, almost no amplification product derived from the sample without DNA purification was confirmed. From these results, it was suggested that taurocholic acid is an inhibitor of DNA amplification.

  Next, using a DNA sample purified from Cryptosporidium oocyst, each bile acid component of taurocholic acid, glycocholic acid, and deoxycholic acid is added to this DNA sample, and then each bile is performed by performing a normal PCR reaction. The effect of acid components on PCR amplification was examined. Extraction and purification of DNA from Cryptosporidium oocysts is performed using the QIAGEN genomic DNA Kit (QIAGEN) according to the supplier's instructions, and 70 mM each of taurocholic acid, glycocholic acid, and deoxycholic acid are added to this purified DNA sample. , X1 / 2, x1 / 10, x1 / 100, x1 / 1000 diluted (corresponding to final concentrations of 10, 5, 1, 0.1, 0.01 mM, respectively), and then PCR reaction under the same conditions ( After performing the primer pair 5′-GGAACCTGGTTGATCCTGCCAG-3 ′; SEQ ID NO: 1 and 5′-GGTCGATCCCCTAACTTTCGTT-3 ′; SEQ ID NO: 2), the amplified products were compared by visualizing them by gel electrophoresis. The result is shown in FIG.

  As the taurocholic acid concentration increased, a marked decrease in the amount of amplified product was confirmed, indicating that taurocholic acid is a strong inhibitor of PCR amplification (DNA polymerase). On the other hand, no change in the amount of amplification product due to the glycocholic acid concentration was confirmed. Further, when the deoxycholic acid concentration was increased, a decrease in the amount of the amplified product was confirmed, but it was not as remarkable as the decrease in the amplified product due to taurocholic acid.

  As described above, since taurocholic acid is a strong inhibitor of PCR reaction, when taurocholic acid is used to oocyst oocysts, the amplification reaction is based on conventional DNA purification techniques. It is necessary to remove taurocholic acid in advance. However, as discussed in Examination Example 1, the conventional DNA purification method causes a decrease in detection sensitivity due to a decrease in DNA extraction efficiency. Therefore, it is preferable to employ it when extracting DNA from Cryptosporidium oocysts. Absent. Therefore, as a method for extracting chromosomal DNA from oocysts, glycocholic acid that can be directly subjected to PCR amplification after decapsulation without going through a DNA purification process and that can achieve high DNA extraction efficiency from oocysts. / Deoxycholic acid treatment / protease treatment, in particular, glycocholic acid treatment followed by protease treatment proved appropriate. The following method for extracting chromosomal DNA from oocysts of Example 2 was performed by glycocholic acid / deoxycholic acid treatment / protease treatment. Here, the reason why a small amount of deoxycholic acid was mixed with glycocholic acid and used for the treatment was to synergistically obtain the respective effects as long as the PCR reaction was not inhibited.

Cryptosporidium species-specific detection was performed as follows. As detection samples, C. parvum and C. muris oocysts purchased from Waterborne (USA) were used, 20 oocysts of each Cryptosporidium were suspended in PBS Buffer, and a total volume of 10 μl environmental sample was prepared. It used for the following experiment. The PBS buffer prepared as x10 PBS Buffer (11.5 g Na ₂ HPO ₄ , 2 g KH ₂ PO ₄ , 80 g NaCl, 2 g KCl / 1 l) was used after diluting at the time of use.

(1) Preparation of chromosomal DNA 4 μl of deoxycholic acid (final concentration 0.1 mM) or glycocholic acid (final concentration 1 mM) was added to each environmental sample and incubated at 37 ° C. for 15 minutes. Subsequently, 0.5 μl proteinase K (600 mAU / ml, manufactured by QIAGEN) and 3.5 μl EDTA (final concentration 10 mM) are added, incubated at 70 ° C. for 10 minutes, and further incubated at 94 ° C. for 10 minutes. Thus, a nucleic acid sample containing chromosomal DNA extracted from oocysts was obtained.

(2) Amplification of DNA The nucleic acid sample prepared above is cooled to room temperature, 2 μl of the sample is collected, and this is used as a template to sense primer primer1 (5′-GGAACCTGGTTGATCCTGCCAG-3 ′; sequence identification number 1), antisense PCR was performed using the primer primer2 (5′-GGTCGATCCCCTAACTTTCGTT-3 ′; SEQ ID NO: 2) as a primer set to obtain an amplified fragment containing the Cryptosporidium gene. The PCR reaction solution is 5 μl of x10 Reaction Buffer (Ex Taq Buffer, manufactured by Takara Bio Inc.), 5 μl of 2.5 mM dNTPs, 1 μl each of 20 pM / μl primer (Primer1, primer2), 5 U / μl Ex Taq (Takara). 0.5 ml, 2 μl of the nucleic acid sample prepared in (1) above and 36 μl of H ₂ O were added to make a total volume of 50 μl. The reaction was performed at 94 ° C for 60 seconds, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds. The reaction was terminated with a final extension reaction of 240 seconds.

(3) Labeling of amplification product 1 μl is collected from the amplification product obtained in (2) above, using this as a template, Cy3 labeled nucleotide as a labeling substance, and sense primer 18Sf60nest (5'-TCAAAGATTAAGCCATGCATGTCT-3 '; SEQ ID NO: 3), antisense primer 18Sr925nest (5'-GATTAATGAAAACATCCTTGGCAAA-3'; SEQ ID NO: 4) is used as a primer set, a second PCR is performed, and a labeled amplification product containing the cryptosporidium gene Got. The PCR reaction solution is 5 μl of X10 Reaction Buffer (Ex Taq Buffer, manufactured by Takara Bio Inc.), 5 μl of 2.5 mM dNTPs, 2 μl of Cy3-dCTP (Pharmacia), and 20 pM / μl of primers (18Sf60nest, 18Sr925nest). 1 μl each, 5 U / μl EX Taq (manufactured by Takara Bio Inc.) 0.5 ml, 1 μl of the amplification product prepared in the above (2) and 81 μl H ₂ O were added to prepare a total volume of 100 μl. The reaction was performed at 94 ° C for 60 seconds, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds, followed by 25 cycles, followed by 72 ° C The reaction was terminated with a final extension reaction of 240 seconds.

(4) Fragmentation of amplification product 100 μl of the labeled amplification product obtained in (3) above was subjected to gel filtration purification using MicroSpin (registered trademark) S-200 HR (Pharmacia), followed by DNase I (Promega To obtain a labeled DNA fragment. DNase treatment was performed as follows. 36 μl was separated from the labeled amplification product after purification by gel filtration, ₄ μl of x10 Reaction Buffer (400 mM Tris-HCl (pH 8.0), 100 mM MgS04, 10 mM CaCl ₂ ) and 0.5 μl of DNase I were added. After incubating at 5 ° C. for 5 minutes, 4 μl of x10 Stop Buffer (20 mM EDTA (pH 8.0)) was added and incubated at 65 ° C. for 10 minutes to inactivate DNaseI. Here, the obtained labeled DNA fragment was subjected to a subsequent hybridization experiment.

(5) Preparation of microarray Poly-L-lysin-coated slide glass (Sigma, USA) was used as a microarray substrate. 20 pmol of each probe shown in Table 1 was mixed in 1.5M Betain and 3X SSC solution, spotted on a slide glass, and irradiated with ultraviolet rays at an intensity of 120 mJ to fix the probe to the substrate. After that, the slide glass was treated in a succinic acid solution (Succinic anhydride 6 g, 1-Methyl-2-pyrrolidinone 325 ml, Sodium borate (1M, pH 8.0) 15 ml) for 10 minutes at room temperature for blocking. . After blocking, it was dehydrated with 100% ethanol for 1 minute, rinsed with distilled water, and dried to prepare a microarray on which the probe was immobilized. Further, as a positive control, a positive control probe (SEQ ID NO: 55) having a consensus sequence of 18S ribosomal gene conserved in all species of Cryptosporidium, and a negative control probe prepared from the sequence of E. coli 16S ribosomal gene as a negative control (Sequence ID No. 56) was immobilized on the microarray in the same manner as described above.

(6) Hybridization Collect 3 μl of the labeled DNA fragment prepared in (4) above, add 6 μl of Hybridization Buffer (12 × SSPE, 2% formamide) and 3 μl of H ₂ O, mix, and mix (5 The probe prepared in (1) was dropped on a microarray fixed on a slide glass. Hybridization is performed by incubating at 45 ° C. for 3 hours, and the slide glass is washed at room temperature. Washing was performed by incubating in 2 × SSC, 0.1% SDS solution for 10 minutes, rinsing 4 to 5 times in 0.2 × SSC, and then centrifuging or completely drying with nitrogen gas. After drying the slide glass, the fluorescence intensity was measured at an excitation wavelength of 550 nm and a fluorescence wavelength of 570 nm using a scanner dedicated to DNA microarray, image processing was performed, and evaluation was performed using an image or a quantitative value of the fluorescence intensity. An image processing diagram is shown in FIG. A, G, C, and T in the figure indicate bases set at the single base mutation site. SSPE and SSC used here were 20 × SSPE (3.6 M NaCl, 0.2 M NaH ₂ PO ₄ , 20 mM EDTA; 210.4 g NaCl, 24.0 g Na H ₂ PO ₄ , 7.4 g EDTA / 1l), 20 × SSC (3 M NaCl, 0.3 M sodium citrate; 88.3 g sodium citrate, 175.4 g NaCl / 1 l) were prepared and diluted before use.

(7) Analysis of results The evaluation is made by calculating the relative fluorescence intensity of each spot with the sum of the fluorescence intensity of the four spots corresponding to the four kinds of probes configured to have four kinds of mutations per base mutation as 100%. Thus, the probe having a relative fluorescence intensity of 60% or more was evaluated as a probe having a complementary sequence of the target gene. FIG. 5 shows the result of specifying complementary sequences based on the image processing diagram of C. parvum shown in FIG.

  In the fifth lane, C. parvum has a relative fluorescence intensity of more than 80% observed in the 5-A spot, and C. muris in the 5-T spot. Parvum is observed in the 8-T spot and C. muris is observed in the 8-A spot with a relative fluorescence intensity of 80% or more. In the 10th lane, C. parvum is 10-T. In C. muris, the relative fluorescence intensity of 80% or more was observed in 10-C spot. Probes corresponding to these spots have complementary sequences to each of C. parvum and C. muris, and the complementary sequences were detected as clearly different from each other. It was confirmed that parvum and C. muris can be accurately identified from the fluorescence pattern. In the present invention, a number of species-specific single nucleotide mutations are specified, and probes having four mutation sites per single nucleotide mutation are disclosed. Therefore, in this example, C. parvum, One skilled in the art will understand that identification of species other than C. muris can be accomplished by similar procedures.

  Further, the target did not react at all with the negative control probe, and strong fluorescence was observed at the spot of the positive control probe. From the above results, it was found that this is an effective method for detecting from the genus to the species level of Cryptosporidium.

[Nucleic acid extraction from Legionella]
DNA was extracted from Legionella pneumophila (L. pneumophila) SG3 strain of Legionella genus and cells of Legionella genus bacteria (Legionella sp.) Of different species by the following procedure, and the presence was confirmed.
(1) Preparation of chromosomal DNA Legionella cells were suspended in PBS to prepare 10 μl of a test sample. To this test sample, 4 μl of reaction solution A (3.5 mM glycocholic acid (final concentration 1 mM), 0.35 mM deoxycholic acid (final concentration 0.1 mM)) was added and incubated at 37 ° C. for 15 minutes. Subsequently, 4 μl of reaction solution B (76.5 mU proteinase K (600 mAU / ml, manufactured by QIAGEN), 45 mM EDTA (final concentration 10 mM)) was added and incubated at 70 ° C. for 10 minutes. Thereafter, proteinase K was inactivated by further incubating at 94 ° C. for 10 minutes to obtain a nucleic acid sample (Example) containing chromosomal DNA extracted from Legionella cells. In addition, the nucleic acid sample which concerns on a comparative example was obtained by processing like an Example except having added the same volume of sterilized water instead of glycocholic acid, deoxycholic acid, proteinase K, and EDTA.

(2) Amplification of DNA The nucleic acid sample and the comparative sample prepared above were cooled to room temperature, 2 μl of each was collected, and PCR was performed using this as a template to obtain an amplified fragment containing the Legionella gene. In the first PCR reaction, sense primer Leg113F (5′-GCACGTGTGTAGCCCTACCC-3 ′; sequence identification number 61) and antisense primer Lrg1115R (5′-ACTGGAACTGAGACACGGTC-3 ′; sequence identification number 62) were used as a primer set. In the second PCR reaction, the sense primer Leg113F and the antisense primer Lrg1115R were used as a primer set. The PCR reaction solution is 5 μl of x10 Reaction Buffer (Ex Taq Buffer, manufactured by Takara Bio Inc.), 5 μl of 2.5 mM dNTPs, 1 μl each of 20 pM / μl primer (Primer1, primer2), 5 U / μl Ex Taq (Takara). 0.5 ml, 2 μl of the nucleic acid sample prepared in (1) above and 36 μl of H ₂ O were added to make a total volume of 50 μl. The reaction was performed at 94 ° C for 60 seconds, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds. The reaction was terminated with a final extension reaction of 240 seconds.

The result of visualizing the amount of DNA extracted by gel electrophoresis is shown in FIG. For each of the L. pneumophila SG3 strain and Legionella sp., And for each of the Examples and Comparative Examples, 3.0 × 10 ⁰ , 3.0 × 10 ¹ , 3.0 × 10 ² , 3.0 × Nucleic acid samples corresponding to 10 ³ , 3.0 × 10 ⁴ cells were loaded into each lane. In the figure, M represents a molecular weight marker of 100 bp ladder. For each of the L. pneumophila SG3 strain and Legionella sp., In the examples where nucleic acid extraction was performed based on this method, gene amplification occurred even from a small number of cells, and the amplified DNA band increased as the number of cells increased. It became thick and quantitative amplification occurred according to the number of cells. This shows that this method can be applied to detection and quantification of Legionella as a quantitative PCR method. On the other hand, when nucleic acid extraction was performed by the method according to the comparative example, a PCR product was produced, but there was no correlation between the number of cells and the thickness of the amplified DNA band, and DNA extraction or PCR reaction was inhibited. There is a risk that.

  Legionella is an intracellularly proliferating gram-negative rod. In protozoa, algae, and the human body, it grows in neutrophils and macrophages, causing pneumonia (legionellosis). Inhabiting a moist environment, it grows in natural water systems such as rivers, as well as in cooling towers and recirculating bathtubs, and human outbreaks of pneumonia may occur. Under such circumstances, in order to detect Legionella rapidly and accurately, it is very useful from an epidemiological viewpoint to extract a nucleic acid using this method for analysis. In particular, legionellosis is often caused by L. pneumophila, and applying this method to nucleic acid extraction of such species can effectively suppress the development of Legionella infection and associated pneumonia in humans. it can.

[Nucleic acid extraction from cultured human cells]
DNA was extracted from cultured human cells (Hela S3) by the following procedure, and its presence was confirmed.
(1) Preparation of chromosomal DNA
Hela S3 cells were suspended in PBS to prepare 10 μl of a test sample. To this test sample, 4 μl of reaction solution A (3.5 mM glycocholic acid (final concentration 1 mM), 0.35 mM deoxycholic acid (final concentration 0.1 mM)) was added and incubated at 37 ° C. for 15 minutes. Subsequently, 4 μl of reaction solution B (76.5 mU proteinase K (600 mAU / ml, manufactured by QIAGEN), 45 mM EDTA (final concentration 10 mM)) was added and incubated at 70 ° C. for 10 minutes. Thereafter, proteinase K was inactivated by further incubating at 94 ° C. for 10 minutes to obtain a nucleic acid sample (Example) containing chromosomal DNA extracted from Hela S3 cells. In addition, the nucleic acid sample which concerns on a comparative example was obtained by processing like an Example except having added the same volume of sterilized water instead of glycocholic acid, deoxycholic acid, proteinase K, and EDTA.

(2) Amplification of DNA The nucleic acid sample prepared above and the comparative sample are cooled to room temperature, 2 μl of each sample is taken out, and this is used as a template to perform nested PCR using primers designed based on the human globin gene. An amplified fragment containing the gene of Hela S3 cell was obtained. In the first PCR reaction, sense primer GH20 (5′-GAAGAGCCAAGGACAGGTAC-3 ′; sequence identification number 63) and antisense primer GH21 (5′-GGAAAATAGACCAATAGGCAG-3 ′; sequence identification number 64) were used as a primer set. In the second PCR reaction, sense primer KM29 (5′-GGTTGGCCAATCTACTCCCAGG-3 ′; sequence identification number 65) and antisense primer PC04 (5′-CAACTTCATCCACGTTCACC-3 ′; sequence identification number 66) were used as a primer set. The PCR reaction solution is 5 μl of x10 Reaction Buffer (Ex Taq Buffer, manufactured by Takara Bio Inc.), 5 μl of 2.5 mM dNTPs, 1 μl each of 20 pM / μl primer (Primer1, primer2), 5 U / μl Ex Taq (Takara). 0.5 ml, 2 μl of the nucleic acid sample prepared in (1) above and 36 μl of H ₂ O were added to make a total volume of 50 μl. The reaction was performed at 94 ° C for 60 seconds, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds. The reaction was terminated with a final extension reaction of 240 seconds.

FIG. 7 shows the result of visualizing the amount of DNA extracted by gel electrophoresis. For each of the Examples and Comparative Examples, nucleic acid samples corresponding to 1 × 10 ⁰ , 1 × 10 ¹ , 1 × 10 ² , 1 × 10 ³ cells were loaded into each lane. In the figure, M represents a molecular weight marker of 100 bp ladder. In the examples in which nucleic acid extraction was performed based on this method, it can be seen that gene amplification occurred even from a small number of cells. On the other hand, when nucleic acid extraction was performed by the method according to the comparative example, a PCR product was generated, but the signal was very weak compared to the example, indicating that the DNA extraction efficiency was poor.

[Nucleic acid extraction from human blood]
DNA was extracted from a human blood sample by the following procedure, and its presence was confirmed.
(1) Preparation of chromosomal DNA Human blood was suspended in PBS to prepare a 10 μl test sample. To this test sample, 4 μl of reaction solution A (3.5 mM glycocholic acid (final concentration 1 mM), 0.35 mM deoxycholic acid (final concentration 0.1 mM)) was added and incubated at 37 ° C. for 15 minutes. Subsequently, 4 μl of reaction solution B (76.5 mU proteinase K (600 mAU / ml, manufactured by QIAGEN), 45 mM EDTA (final concentration 10 mM)) was added and incubated at 70 ° C. for 10 minutes. Thereafter, proteinase K was inactivated by further incubating at 94 ° C. for 10 minutes to obtain a nucleic acid sample (Example) containing chromosomal DNA extracted from human blood. In addition, the nucleic acid sample which concerns on a comparative example was obtained by processing like an Example except having added the same volume of sterilized water instead of human blood.

(2) Amplification of DNA The nucleic acid sample prepared above and the comparative sample are cooled to room temperature, 2 μl of each sample is taken out, and this is used as a template to perform nested PCR using primers designed based on the human globin gene. An amplified fragment of a gene contained in human blood was obtained. In the first PCR reaction, sense primer GH20 (5′-GAAGAGCCAAGGACAGGTAC-3 ′; sequence identification number 63) and antisense primer GH21 (5′-GGAAAATAGACCAATAGGCAG-3 ′; sequence identification number 64) were used as a primer set. In the second PCR reaction, sense primer KM29 (5′-GGTTGGCCAATCTACTCCCAGG-3 ′; sequence identification number 65) and antisense primer PC04 (5′-CAACTTCATCCACGTTCACC-3 ′; sequence identification number 66) were used as a primer set. The PCR reaction solution is 5 μl of x10 Reaction Buffer (Ex Taq Buffer, manufactured by Takara Bio Inc.), 5 μl of 2.5 mM dNTPs, 1 μl each of 20 pM / μl primer (Primer1, primer2), 5 U / μl Ex Taq (Takara). 0.5 ml, 2 μl of the nucleic acid sample prepared in (1) above and 36 μl of H ₂ O were added to make a total volume of 50 μl. The reaction was performed at 94 ° C for 30 minutes, followed by 30 cycles of denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds. The reaction was terminated with a final extension reaction of 240 seconds.

The results of visualizing the amount of DNA extracted by gel electrophoresis are shown in FIG. For the examples, nucleic acid samples corresponding to 1 × 10 ² , 1 × 10 ³ , 1 × 10 ⁴ , 1 × 10 ⁵ cells (total number of red blood cells, white blood cells, and platelet cells) were loaded into each lane. The rightmost lane is a nucleic acid sample (0 cells) according to the comparative example. In the figure, M represents a molecular weight marker of 100 bp ladder. In the examples in which nucleic acid extraction was performed based on this method, it can be seen that gene amplification occurred even from a small number of cells. And since the amplified fragment was not obtained from the nucleic acid sample which concerns on a comparative example, it turns out that this amplification is specific.

  As is apparent from the above-described two embodiments, nucleic acid extraction from human cells and blood and subsequent analysis can be performed efficiently by employing this method. Therefore, when collecting cells for treatment or the like, the amount of collection is small, so that the burden on the patient can be reduced. Moreover, a rapid and accurate diagnosis can be performed by applying this method to nucleic acid extraction from human cells and blood.

[Identification of microorganisms using microarrays]
Probes for specifying microorganisms to be detected were fixed as 8 × 24 spots in a 3 mm × 8 mm square region on a carrier (Poly-L-lysin coated glass slide (Sigma, USA)). As shown in FIG. 9 (a), 4 × 12 probe spots for detecting the E. coli gene prepared on the basis of the Flagellin gene sequence of E. coli, and the dehalogenase of sulfate-reducing bacteria. Created based on a conserved region (OWP gene sequence) common to all Cryptosporidium, 4 x 12 probe spots for detecting a sulfate-reducing bacteria gene capable of assimilating trichlorethylene made based on the gene sequence 4 x 4 probe spots for detecting Cryptosporidium gene, 4 spots for detecting Cryptosporidium gene of the type created based on Cryptosporidium 18S ribosomal gene sequence There are 20 pieces.

  Nucleic acid extraction method according to the present invention from each test sample containing Escherichia coli (gram-negative bacteria), sulfate-reducing bacteria having a dehalogenase gene (gram-positive bacteria), and multiple types of cryptostridium (gram-negative bacteria) ( Chromosomal DNA was extracted using glycocholic acid / deoxycholic acid treatment / protease treatment). PCR was carried out using these nucleic acid samples as templates, and each amplification product obtained was further subjected to PCR in a system containing labeled nucleotides to obtain labeled amplification products. These were fragmented with DNase I (Promega) to obtain labeled DNA fragments. These labeled DNA fragments were added (dropped) to the microarray and incubated to perform hybridization, and the slide glass was washed at room temperature. After drying the slide glass, the fluorescence intensity was measured with a dedicated scanner for DNA microarray, image processing was performed, and evaluation was performed using the image or the quantitative value of the fluorescence intensity. Image processing diagrams are shown in FIGS.

  FIG. 9B is an image processing diagram when a labeled DNA fragment derived from sulfate-reducing bacteria is added to the microarray. Strong fluorescence was generated only from a spot corresponding to a probe having a sequence derived from a dehalogenase gene of a sulfate-reducing bacterium, and such a microorganism could be detected. FIG. 9C is an image processing diagram when a labeled DNA fragment derived from E. coli is added to the microarray. Strong fluorescence was generated only from a spot corresponding to a probe having a sequence derived from the flagellin gene of E. coli, and such a microorganism could be detected. FIG. 10 (a) is an image processing diagram when a plurality of types of labeled DNA fragments derived from Cryptosporidium are added to the microarray. Spots corresponding to the above-mentioned sequences derived from the 18s rDNA gene of multiple types of Cryptosporidium (each column is composed of 4 types of probes that detect single nucleotide polymorphisms characterizing each type of Cryptosporidium) Corresponding to the pattern of the mold, strong fluorescence was generated only for one of the probes forming the column, and it was possible to clearly detect and identify each type.

  FIG. 10 (b) shows that a chromosomal DNA is extracted from a test sample in which cryptostridium and E. coli are mixed according to this method, and a labeled DNA fragment is obtained in the same manner as described above, and this is added to the microarray. It is the result. Strong fluorescence was generated only from spots corresponding to probes having a sequence derived from a conserved region (OWP gene sequence) common to all Cryptosporidium and probes having a sequence derived from the flagellin gene of Escherichia coli. Each microorganism could also be detected from the test sample.

  FIG. 10 (c) shows a method for extracting chromosomal DNA from a test sample obtained by mixing sulfate-reducing bacteria having a dehalogenase gene and E. coli according to this method, and obtaining a labeled DNA fragment in the same manner as described above. It is the result of adding to the microarray. Strong fluorescence is generated only from spots corresponding to the probe having a sequence derived from the dehalogenase gene of sulfate-reducing bacteria and the probe having a sequence derived from the flagellin gene of Escherichia coli. Microbe detection was possible. It was also found that this method can be applied to both gram-negative and positive bacteria.

Electrophoretic diagram showing the results of Study Example 1 comparing the DNA extraction method of the present invention with the conventional method Electrophoretic diagram showing the results of Study Example 2 in which the effects of taurocholic acid, deoxycholic acid, and glycocholic acid on PCR amplification were examined. Electrophoretic diagram showing the results of Study Example 2 in which the effects of taurocholic acid, deoxycholic acid, and glycocholic acid on PCR amplification were examined. Fluorescence intensity image processing diagram showing the results of species-specific detection of C. parvum and C. muris by the method of the present invention The figure which shows the result of having specified the complementary arrangement | sequence based on the image processing figure of C. parvum of FIG. Electrophoretic diagram showing the results of comparing the DNA extraction method of the present invention with the conventional method using Legionella cells as the test sample Electrophoresis diagram showing the results of comparing the DNA extraction method of the present invention with the conventional method using human cultured cells as the test sample Electrophoretic diagram showing the results of using the DNA extraction method of the present invention using human blood as a test sample Image processing diagram of fluorescence intensity showing the result of detection of various microorganisms by the method of the present invention Image processing diagram of fluorescence intensity showing the result of detection of various microorganisms by the method of the present invention

Claims (9)

  A nucleic acid extraction method comprising a step of bringing glycocholic acid into contact with a test sample.   The nucleic acid extraction method according to claim 1, wherein the concentration of glycocholic acid is 1 to 10 mM.   The nucleic acid extraction method according to claim 1, further comprising a step of bringing a proteolytic enzyme into contact with the test sample.   The nucleic acid extraction method according to claim 1, wherein the test sample is a sample derived from an animal.   The nucleic acid extraction method according to claim 1, wherein the test sample is a sample containing microorganisms.   The nucleic acid extraction method according to claim 5, wherein the microorganism is Cryptosporidium.   A nucleic acid extraction kit containing glycocholic acid.   The nucleic acid extraction kit according to claim 7, comprising glycocholic acid so that the final concentration is 1 to 10 mM at the time of use.   The nucleic acid extraction kit according to claim 7 or 8, comprising a proteolytic enzyme.