JP2010029068A - Method for producing inner standard dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an inner standard DNA suitably used for confirming a PCR reaction by a hybridization method, especially by which the inner standard DNA capable of being amplified with a primer for an objective DNA and not hybridizing with a probe for the objective DNA can efficiently be produced. <P>SOLUTION: There is provided the method for producing the inner standard DNA which is added to a reaction solution of a PCR reaction for amplifying the objective DNA originated from a detection target bacterium and is amplified with a primer used for the objective DNA and amplifying the objective DNA, characterized by selecting an inner standard candidate DNA satisfying prescribed conditions by a data base retrieval, and converting the sequences of the 5'-terminal domain and 3'-terminal domain of the inner standard candidate DNA to the same sequences as the forward direction primer 21 sequence and backward direction primer 22 complementary sequence of the primer for the objective DNA, by a PCR method using a substituted primer. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a so-called internal standard DNA.

  The PCR method is widely used as a means for amplifying DNA, and a method of adding an internal standard to the reaction solution is known in order to confirm that the PCR reaction is being performed reliably. For example, by adding an internal standard DNA and a primer for amplifying it to a PCR reaction solution, and confirming the amplification of the internal standard DNA after the PCR reaction, it can be confirmed that the PCR reaction has been performed correctly. . The amplification of the internal standard DNA is confirmed by, for example, performing an electrophoresis method on the amplification product and confirming a band indicating the internal standard DNA at a predetermined position. Alternatively, for example, a DNA microarray in which an internal standard probe that hybridizes with the internal standard DNA is used, and hybridization between the internal standard DNA and the probe is confirmed by fluorescence.

  As described above, the internal standard DNA is added to a solution subjected to the PCR reaction and used as a positive control for confirming that the reaction has been appropriately performed. Specifically, in order to identify the causative bacteria of an infectious disease, a PCR reaction is performed on a DNA solution prepared from a specimen (for example, urine or blood) using a primer that amplifies the DNA encoding the causative 16s rRNA. Do. At this time, an internal standard DNA and an internal standard primer (primer that specifically amplifies the internal standard DNA) are added, and a PCR reaction is performed. When a hybridization reaction is performed on the solution after the PCR reaction using a DNA microarray on which an internal standard probe (probe that specifically binds to the internal standard DNA) is immobilized, the PCR reaction is appropriately performed. The internal standard DNA and the internal standard probe react to emit fluorescence. Therefore, even if the causative bacterium is negatively determined, it can be confirmed that the PCR reaction has been performed correctly.

  As a DNA microarray for identifying the causative bacteria of an infectious disease, for example, Patent Document 1 discloses a DNA microarray for detecting infectious disease-causing bacteria. In order to detect causative bacteria, this DNA microarray is based on a probe having a base sequence that specifically binds to DNA encoding 16s rRNA of causative bacteria and an internal standard probe. Probes that target each causative bacterium are designed, for example, from the genome portion coding for 16s rRNA, and are designed to be highly specific to the causative bacterium and to expect sufficient hybridization sensitivity. .

  The internal standard DNA is used not only as a positive control but also for examining DNA having a target base sequence contained in an unknown sample. For example, a known amount of internal standard DNA is added to a sample collected from a patient, the internal standard DNA is amplified simultaneously with the target DNA amplification reaction, and the amplification rate of the target DNA is calculated from the amplification rate of the internal standard DNA. An estimation method is included. If the internal standard DNA to be added is selected to be close to the size of the target DNA, the concentration of the target DNA can be quantified. In particular, more accurate quantification can be achieved by performing PCR reaction by diluting the internal standard DNA stepwise. In addition, the comparison of the concentration in the amplified product can also be performed by comparing the density of the bands by electrophoresis, but it is preferable to compare the fluorescence development using a DNA microarray because a large number of samples can be measured at once. it is conceivable that.

  However, in the quantitative analysis method of the target DNA using the internal standard DNA by the PCR method, the reliability of the quantitative and reproducibility may be lowered due to the difference in the amplification efficiency and uncertainty of the target DNA and the internal standard DNA. is there. That is, in order to amplify the internal standard DNA, an internal standard primer is added to the reaction solution. However, since the hybridization efficiency of the target DNA differs from that of the target DNA primer, the internal standard is different. A difference occurs in the amplification efficiency between the DNA and the target DNA, which is the reason for affecting accurate quantification. Since the amplification reaction proceeds exponentially in the PCR reaction, the quantitative error becomes large even if the difference in the hybridization efficiency is small.

  In order to reduce the quantification error, in Patent Document 2, the internal standard DNA is used by using the target DNA primer by setting the 5 ′ end and the 3 ′ end of the internal standard DNA in common with the base sequence of the target DNA. Can be amplified. In addition, the difference in hybridization efficiency can be reduced by designing the internal standard DNA in this way. Furthermore, it is not necessary to add an internal standard primer.

JP 2004-3131181 A Japanese Patent No. 3331178

  As described above, when the target DNA is quantified using the internal standard DNA, the 5 ′ end and the 3 ′ end of the internal standard DNA can be used as a sequence common to the base sequence of the target DNA and amplified using the target DNA primer. It is desirable to design the internal standard DNA. However, Patent Document 2 only discloses a specific internal standard DNA used for quantification of a specific target DNA, and does not describe a method for producing the internal standard DNA. Further, there is no description of an internal standard DNA useful when confirmation or quantification is performed by a hybridization method using a DNA microarray.

  Accordingly, an object of the present invention is to provide a method for producing an internal standard DNA that is suitably used for confirming the PCR reaction by a hybridization method. In particular, an object of the present invention is to provide an efficient method for producing an internal standard DNA that can be amplified with a target DNA primer and that does not hybridize to a target DNA probe.

The method for producing an internal standard DNA according to the present invention includes:
A method for producing an internal standard DNA that is added to a reaction solution of a PCR reaction for amplifying a target DNA derived from a detection target bacterium and amplified by a target DNA primer for amplifying the target DNA,
Select internal standard candidate DNA satisfying the following conditions by database search,
The sequences of the 5 ′ terminal region and 3 ′ terminal region of the internal standard candidate DNA are the same as the complementary sequences of the forward primer sequence and the reverse primer of the target DNA primer, respectively, by PCR using a replacement primer. It is characterized by.
Condition (A): a sequence partially including a region having a predetermined homology with the forward primer (hereinafter abbreviated as region of Condition A).
Condition (B): a sequence partially including a region having a predetermined homology with a complementary sequence of the reverse primer (hereinafter abbreviated as a region of Condition B).
Condition (C): a sequence in which the region of Condition A is on the 5 ′ end side and the region of Condition B is on the 3 ′ end side.
Condition (D): The sequence between the region of the condition A and the region of the condition B has the same number of bases as the target DNA, and has low homology with the 16S rRNA sequence of the detection target bacteria.

  According to the present invention, it is possible to easily produce an internal standard DNA that is suitably used for confirming a PCR reaction by a hybridization method.

  In this specification, the target DNA is a DNA to be amplified by a PCR reaction, and is a DNA having a sequence of one region in 16S rRNA of a detection target fungus. The target DNA primer is a primer used to amplify the target DNA. Although the following explanation is not intended to be particularly limited, for example, the detection of microorganisms and bacteria is performed using DNA extracted from a test sample (urine or blood) containing the bacteria as a template. This is performed by amplifying the target DNA by a PCR reaction using a primer (primer for the target DNA) designed from a specific sequence. Specifically, for example, infectious disease-causing bacteria can be detected by designing a primer specific to the causative bacteria from DNA encoding 16S rRNA and collecting a test sample (eg, urine or blood) collected from a patient or the like. PCR is carried out based on the DNA contained in the DNA, and the amplification of the target DNA (part of 16S rRNA) is confirmed. When there are a plurality of bacteria to be detected, primers specific to all the bacteria are prepared based on the DNA sequence encoding 16S rRNA of those bacteria.

  As described above, in general, primers specific to the causative fungus are designed from the DNA portion encoding 16S rRNA. FIG. 1 is a schematic diagram showing the positional relationship between DNA (11) encoding 16S rRNA in DNA extracted from, for example, a causative bacterium, and DNA (target DNA) (12) amplified by a target DNA primer. . Further, in FIG. 2, the target DNA primers (21, 22) for amplifying the target DNA (12) are shown. Among the target DNA primers, the primer that advances DNA synthesis in the forward direction is the forward primer (21), and the primer that advances DNA synthesis in the reverse direction is the reverse primer (22). FIG. 3 shows the target DNA to be amplified in the PCR reaction. In this specification, the target DNA portion corresponding to the portion to which the forward primer (21) binds is referred to as the 5 ′ end region (31) of the target DNA, and the portion to which the reverse primer (22) binds. The target DNA portion corresponding to is called the 3 ′ end region (33) of the target DNA. The target DNA portion between the 5 'end region (31) of the target DNA and the 3' end region (33) of the target DNA is referred to as the central region (32) of the target DNA.

  Although DNA which has target DNA is not specifically limited, For example, it can prepare by extracting from biological samples, such as blood, by an appropriate method. The presence or absence of amplification of the target DNA is not particularly limited. For example, a DNA microarray on which a probe for the target DNA that specifically binds to the target DNA is immobilized, shows a positive labeling signal in the hybridization reaction. It is confirmed by whether or not.

  Next, the internal standard DNA produced by the production method according to the present invention will be described.

  FIG. 4 is a conceptual diagram showing an internal standard DNA produced by the present invention. In FIG. 4 (a), (41) is the 5 'end region of the internal standard DNA, and (42) is the 3' end region. The region between the 5 'end region and the 3' end region is referred to as the central region (43). The 5 'end region is a region corresponding to a portion to which a forward primer is attached in the target DNA primer, and the 3' end region is a region corresponding to a portion to which a reverse primer is attached. Here, the sequence of the 5 'end region of the internal standard DNA is the same sequence as the forward primer sequence, and the sequence of the 3' end region of the internal standard DNA is the same sequence as the complementary sequence of the reverse primer. For this reason, the internal standard DNA is amplified by the forward primer and the reverse primer. That is, this internal standard DNA is simultaneously amplified when the target DNA is amplified by a PCR reaction using the target DNA primer. Further, the internal standard DNA has a low homology with the 16s rRNA sequence of the detection target bacterium. The length of the sequence of the internal standard DNA is preferably as close as possible to the length of the target DNA.

  The internal standard DNA having the above characteristics can be amplified with primers for the target DNA during the PCR method, and the internal standard DNA can be detected without reacting with the probe for detecting the causative bacteria during the hybridization reaction of the DNA microarray. It can be detected with a probe. By measuring the luminance of the internal standard DNA detection probe, PCR amplification assay can be performed. Further, by comparing the measurement brightness of the internal standard DNA detection probe with the brightness of the causative bacteria detection probe, the quantitativeness can be improved. By designing the internal standard DNA in this way, the internal standard DNA can be amplified with the target DNA primer, and the internal standard DNA does not bind to the target DNA probe during the hybridization reaction. .

Here, the method for producing the internal standard DNA according to the present invention is as follows.
A method for producing an internal standard DNA that is added to a reaction solution of a PCR reaction for amplifying a target DNA derived from a detection target bacterium and amplified by a target DNA primer for amplifying the target DNA,
Select internal standard candidate DNA satisfying the following conditions by database search,
The sequences of the 5 ′ terminal region and 3 ′ terminal region of the internal standard candidate DNA are the same as the complementary sequences of the forward primer sequence and the reverse primer of the target DNA primer, respectively, by PCR using a replacement primer. It is characterized by.
Condition (A): a sequence partially including a region having a predetermined homology with the forward primer (hereinafter abbreviated as region of Condition A).
Condition (B): a sequence partially including a region having a predetermined homology with a complementary sequence of the reverse primer (hereinafter abbreviated as a region of Condition B).
Condition (C): a sequence in which the region of Condition A is on the 5 ′ end side and the region of Condition B is on the 3 ′ end side.
Condition (D): The sequence between the region of the condition A and the region of the condition B has the same number of bases as the target DNA, and has low homology with the 16S rRNA sequence of the detection target bacteria.

  Hereinafter, the method for producing the internal standard DNA according to the present invention will be described.

  FIG. 5 is a flowchart showing a procedure for producing an internal standard DNA by the production method according to the present invention. The method for producing an internal standard DNA according to the present invention mainly comprises a step of selecting an internal standard candidate DNA to be a template of the internal standard DNA and a step of replacing the mismatch site of the selected internal standard candidate DNA by the PCR method. Divided. Hereinafter, the selection of the internal standard candidate DNA and the sequence replacement step of the mismatch site will be described in detail.

(Selection of internal standard candidate DNA)
Analyzes of gene sequences are partly underway, and information on these already analyzed gene sequences is registered in a database and provided so that it can be used in common. General researchers can use information on gene sequences by accessing such a database. Such a gene sequence database is not particularly limited, and examples thereof include gene databases such as GenBank in the United States, EMBL in Europe, and the National Institute of Genetics in Japan.

In the present invention, first, an internal standard candidate DNA is selected by database search, a mutation is introduced into the selected internal standard candidate DNA, and an internal standard DNA that can be amplified with a target DNA primer is prepared. This internal standard candidate DNA is obtained by searching for the following sequences.
Condition (A): a sequence partially including a region having a predetermined homology with the forward primer (hereinafter abbreviated as region of Condition A).
Condition (B): a sequence partially including a region having a predetermined homology with the complementary strand of the reverse primer (hereinafter abbreviated as the region of Condition B).
Condition (C): The region of condition A is on the 5 ′ end side, and the region of condition B is on the 3 ′ end side.
Condition (D): The sequence between the region of condition A and the region of condition B is the same as the number of bases of the target DNA, and has low homology with the 16S rRNA sequence of the fungus to be detected.

  Such selection of the internal standard candidate DNA can be performed, for example, by the following method. The following selection method is an example, and the method for selecting sequence data that satisfies the above conditions (A) to (D) is not limited to the following selection method.

  First, each time a predetermined search is performed, the user selects one database, selects a file to be searched, and specifies various parameters such as scores such as homology. As a file to be searched, gene information of a plurality of species may be selected, or gene information of one type of species may be selected.

  Next, using the selected data file, sequence data partially including a sequence having a predetermined homology with the forward primer is searched. This makes it possible to search for a sequence having homology with the forward primer. Here, the homology score is preferably set to 50% or more, more preferably set to 70% or more, and further preferably set to 80% or more. Then, DNA information having a hit sequence in the search for the forward primer is selected (DNA information (1)).

  Next, using the DNA information (1), sequence data partially including a region having homology with the complementary strand of the reverse primer is searched. This makes it possible to search for a sequence having homology with the complementary sequence of the reverse primer. Here, the homology score is preferably set to 50% or more, more preferably set to 70% or more, and further preferably set to 80% or more. Then, DNA information having a sequence hit in the reverse primer search is selected (DNA information (2)).

  Next, in the DNA information (2), sequence data in which the condition A region is on the 5 ′ end side and the condition B region is on the 3 ′ end side is selected. Further, sequence data having the same number of bases from the condition A region to the condition B as the number of bases of the target DNA is selected (the information of the sequence data is DNA information (3)). The same level is not particularly limited, but the difference between them is preferably 50 bases or less, more preferably 20 bases or less, and even more preferably 10 bases or less.

  Next, in the DNA method (3), a sequence having a low homology with the 16S rRNA sequence of the detection target bacterium is selected as the internal standard candidate DNA. For example, the homology score is preferably 50% or less, more preferably 20% or less, and even more preferably 10% or less. In addition, when there are multiple types of detection target bacteria, the “16S rRNA sequence of the detection target bacteria” is a sequence having high homology to the 16S rRNA sequences of all the detection target bacteria. The “16S rRNA sequence of the detection target bacterium” preferably has 80% or more homology with all 16S rRNA sequences of the detection target bacterium, and more preferably 90% or more.

  Note that the search order is not particularly limited.

  Further, as the internal standard candidate DNA, it is preferable to select a higher-order one having a small number of bases in the mismatch site. As a selection condition at this time, the smaller the number of bases in the mismatch site, the better. The mismatch site is replaced with the correct sequence in the sequence replacement step described later, but in order to reduce the replacement step, as a selection criterion, ensure a homology of 50% or more of the total number of bases of the corresponding forward and reverse primers. It is desirable that Further, the difference in the number of mismatches between the 5 'end and the 3' end is preferably 5 bases or less.

  Moreover, it is preferable that the Tm value of the internal standard DNA to be selected is approximately the same as the Tm value of the target DNA. Specifically, the difference between the Tm values is preferably 5 ° C. or less, and more preferably 3 ° C. or less.

The internal standard DNA is added to the PCR solution. However, when the target DNA is detected by the hybridization method after the PCR reaction, it is necessary that the internal standard DNA does not react with the target DNA probe. When preparing such an internal standard DNA, the internal standard candidate DNA to be selected should not have a sequence between the region of condition A and the region of condition B that hybridizes with the target DNA probe. . Therefore, in addition to the conditions (A) to (D), a condition (E); a sequence that does not have a sequence that hybridizes with the target DNA probe is added between the region of the condition A and the region of the condition B. Is preferred. In order to confirm that there is no hybridizing sequence, for example, the sequence of the target DNA probe is not present in the sequence of the internal standard candidate DNA, and one or several bases are substituted in the sequence of the target probe. What is necessary is just to confirm with DNA data that there exists no sequence. At this time, it may be confirmed that it does not hybridize to a complementary strand with the sequence represented on the data. Specifically, when the target probe is an oligonucleotide of 20 bases, if this sequence and the sequence of the internal standard candidate DNA have 85% or less homology, there is a difference of 3 bases or more, They are mismatched with each other and do not hybridize. The homology conditions can be determined based on the conditions of the hybridization reaction.
The “target DNA probe” is a probe containing a polynucleotide that can specifically hybridize to the DNA of the detection target bacterium. The length of the target DNA probe is, for example, 10 to 200 nucleotides, preferably 20 to 100 nucleotides, more preferably 40 to 80 nucleotides. Further, there may be only one target DNA probe or a plurality of target DNA probes. When a plurality of DNAs are used, an internal standard candidate DNA that does not hybridize to all the target DNA probes is selected.

(Sequence replacement process of mismatch site)
Next, DNA is extracted from the biological species having the selected internal standard candidate DNA. DNA extraction can be performed by a conventionally known method, and is not particularly limited.

  Next, the mismatch base between the target DNA primer and the internal standard candidate DNA is replaced with a correct base using a PCR reaction using a replacement primer.

  As shown in FIG. 6, when there are a plurality of mismatch sites, PCR amplification is first performed with the first substitution primer that replaces the first region, and the mismatch site of the internal standard candidate DNA is used as the first primer. Convert to complementary sequence. Similarly, PCR amplification is performed on the sequence obtained by the substitution process using a second substitution primer for substituting the second region, and the mismatch site is converted into a sequence complementary to the second primer. By repeating these processes until there are no mismatch sites, all mismatch sites in the internal standard candidate DNA are eliminated, and an internal standard DNA having a sequence complementary to the target DNA primer is obtained.

(Embodiment 1)
Hereinafter, embodiments of the present invention will be described, but the present invention is not particularly limited to the following embodiments.

“Selection of internal standard candidate DNA”
First, selection of the internal standard candidate DNA will be described.

  For example, as shown in FIG. 5, using a sequence database in which a large number of base sequences are registered, a sequence partially including a sequence having homology with a forward primer is searched (501). In addition, for example, SSEARCH can be used as the homology search program, and a data set obtained by extracting a data set related to Arabidopsis mitochondria (searched with “A thaliana” or “mitochondrial”) from NCBI as the sequence database can be used. The result list of the homology search is used as a condition for selecting an internal standard candidate DNA.

  Next, the sequence database is searched for a sequence partially including a sequence having homology with the complementary strand of the reverse primer (502). This homology search result list is also used as a condition for selecting an internal standard candidate DNA.

  Next, using a sequence having high homology as compared with the 16s rRNA sequences of all causative bacteria to be detected, homology is obtained with respect to the sequence between the condition A region and the condition B region of the sequence database. Check (503).

  Next, an internal standard candidate DNA is selected (504). Here, using the result list of 501, 502, and 503, candidate DNA that satisfies all of the following conditions is selected.

(1) It has a region having high homology with the forward primer on the 5 ′ end side.
(2) It has a region having high homology with the reverse primer on the 3 ′ end side.
(3) The homology between the 16s rRNA of the detection target bacterium and the sequence between the condition A region and the condition B region is low.
(4) The difference between the number of bases between the condition A region and the condition B region and the number of bases of the target DNA is within 50 bases.

  The above conditions (1) and (2) are selected from the result list of the homology search of 501 and 502, and the upper one with a small number of bases in the mismatch site. As a selection condition at this time, the smaller the number of mismatched bases, the better. The mismatch sequence can be replaced with the correct sequence by the process described later. However, in order to reduce the sequence replacement step, the total of the corresponding forward and reverse primers is used as a selection criterion for both the sequences (1) and (2). It is desirable to ensure homology of 50% or more of the number of bases. The condition (3) above selects those that did not appear in the list of 303 homology searches (that did not hit in the SSEARCH search). It is even better if the length / Tm value of the sequence in the central region sandwiched between the forward and reverse primers is comparable to the length / Tm value of the 16s rRNA sequence. A sequence satisfying all the above conditions (1) to (4) is an internal standard candidate DNA.

  FIG. 7 shows the alignment of DNA data (X98301) selected as an internal standard candidate DNA and forward / reverse primer using the above conditions.

"Sequence replacement process"
Next, a specific procedure for the sequence replacement process at the mismatch site of the internal standard candidate DNA will be described. First, a primer for substituting one region of mismatch of the internal standard candidate DNA is designed (505) (see FIG. 5). Only one mismatch region is made the same as the primer sequence of 16s rRNA, and other mismatch regions are made the same sequence as the internal standard candidate DNA. In consideration of the success rate of PCR amplification, the length of the mismatch region to be substituted at one time is desirably suppressed to 2 bases or less. Next, PCR amplification is performed using the above primers to obtain an internal standard candidate DNA in which the sequence of one region is substituted (506). The above processes 505 and 506 are repeated until there is no mismatch area (507). By performing the above processing, the internal standard DNA is completed (508).

  FIG. 8 shows an example in which the sequence replacement process of the mismatch region is performed on the region of the forward primer of X98301. A primer for substituting the mismatch region one by one is designed and replaced with an internal standard DNA having the same sequence as the 16s rRNA amplification primer by four PCR amplifications.

  FIG. 15 shows an internal standard DNA sequence prepared by the above-described internal standard DNA manufacturing method based on the DNA data X98301. The sequence from 1031 to 2739 of X98301 is replaced with the same sequence as the complementary sequence of the forward primer and the reverse primer at the 5 'end and 3' end, respectively.

  FIG. 9 is a block diagram showing a configuration of an information processing apparatus to which a method for searching a sequence database is applied. The database search system is mounted on a device including an external storage device 901, a central processing unit (CPU) 902, a memory 903, and an input / output device 904. The external storage device 901 holds the luminance level as a result of the program for realizing the present invention and the hybridization reaction. A central processing unit (CPU) 902 executes a program that implements the present invention and controls all devices. The memory 903 temporarily records programs used by the central processing unit (CPU) 902 and data such as subroutines and array data. The input / output device 904 interacts with the user. In many cases, the user triggers program execution via this input / output device. The user's result confirmation and program parameter control are performed via this input / output device.

  An external storage device 901 as a gene database is connected to the CPU 902 here, and gene sequence data is obtained from a storage medium such as a CD-ROM or a hard disk in which sequence information of gene information is stored in the external storage device 901. be able to. Then, a homology search is performed on the sequence for searching for homology. Further, the CPU 902 can connect to a database as another gene database via a network. In this case, the information of the gene database is obtained from the database, and the homology search is performed.

  FIG. 10 is a diagram illustrating a configuration example of a fluorescence image capturing apparatus that captures the result of a hybridization reaction of a DNA microarray. The fluorescence image pickup apparatus includes the information processing apparatus 1001 shown in FIG. 7, an image pickup apparatus 1003 with a built-in CMOS sensor 1004 and a fluorescent filter 1005, a DNA microarray excitation light source 1006, a DNA microarray support base 1008 that supports the DNA microarray 1007, It is composed of a dark box 1002 that blocks light from the outside. The CMOS sensor 1004 is equipped with an RGB color filter. The microarray 1007 that has undergone the hybridization reaction is excited by an excitation light source 1006, and the emitted fluorescence is imaged by the imaging device 1003 through the fluorescence filter 1005. When the Cy3 fluorescent dye is used, the excitation light source 1006 uses a laser having a wavelength of 532 nm. The imaging device 1003 is operated from the input / output device of the information processing device 1001 connected by a USB cable.

  FIG. 11 is a diagram showing the state of hybridization on the DNA microarray. In most cases, DNA has a double helix structure, and the bond between the two strands is realized by hydrogen bonding between bases. On the other hand, RNA often exists in a single strand. The types of bases are 4 types of ATGC in the case of DNA and 4 types of AUGC in the case of RNA, and the base pairs capable of hydrogen bonding are AT (U) and GC pairs, respectively. In general, a hybridization reaction refers to a state in which single-stranded nucleic acid molecules are partially bound to each other through a partial base sequence in the nucleic acid molecule. .

  Next, the overall operation procedure using the DNA microarray will be described with reference to FIG. The “sample” of 1201 is a liquid or individual that should contain the target nucleic acid (target DNA). For example, when the present invention is applied to identify the causative bacteria of infectious diseases, such as blood derived from animals such as humans and livestock, body fluids such as sputum, gastric juice, vaginal secretions, oral mucus, urine and feces Everything that seems to contain bacteria, such as effluent, becomes a sample. In addition, a medium that may cause contamination by bacteria, such as food poisoning, food subject to contamination, drinking water, and water in the environment such as hot spring water, may be used as a sample. In addition, animals and plants such as quarantine at the time of import / export are also subject to this. The “internal standard DNA” 1202 is prepared using the internal standard DNA production method according to the present invention.

  Next, the target DNA and 1202 internal standard DNA contained in the sample 1201 are simultaneously amplified using the “biochemical amplification” method 1203. For example, when the present invention is applied to identify the causative bacteria of infectious diseases, the target DNA and internal standard DNA are amplified by the PCR method using a PCR reaction primer designed for detecting 16s rRNA as a target DNA primer. can do. Alternatively, a PCR reaction or the like is further performed based on the PCR amplification product. Moreover, you may adjust by amplification methods, such as LAMP method other than PCR.

  Thereafter, the amplified target DNA and internal standard DNA are labeled by various labeling methods for visualization. As this labeling substance, fluorescent substances such as Cy3, Cy5 and Rhodamin are usually used. Further, in the experimental procedure of biochemical amplification of 1203, a labeled molecule may be mixed.

  Then, the nucleic acid to which the labeled molecule has been added undergoes a hybridization reaction (1206) with 1205 DNA microarray. This situation is as shown in FIG. For example, when the present invention is applied to identify a causative bacterium of an infectious disease, the DNA microarray 1205 includes a target DNA probe having a base sequence specific to the causative bacterium and a base sequence specific to the internal standard DNA. The probe having the is fixed to the substrate. The design of the probe of each bacterium is, for example, highly specific to the bacterium than the genome part coding 16s rRNA, and has a hybridization sensitivity that is sufficient and does not vary as much as possible with each probe base sequence. Do as you expect. The carrier (substrate) for fixing the probe of the DNA microarray 1205 may be a flat substrate such as a glass substrate, a plastic substrate, or a silicon wafer. Further, even if a three-dimensional structure having irregularities, a spherical shape such as a bead, a rod shape, a string shape, a thread shape, or the like is used, the embodiment and the effect of the present invention are not affected.

  Usually, the surface of the substrate is treated so that the probe DNA can be immobilized. In particular, a product having a functional group introduced so that a chemical reaction is possible on the surface is a preferable form in terms of reproducibility because the probe is stably bound in the course of the hybridization reaction. Examples of the immobilization method include an example using a combination of a maleimide group and a thiol (—SH) group. That is, a thiol (-SH) group is bonded to the end of the nucleic acid probe, and the solid phase surface is treated so as to have a maleimide group, so that the thiol group of the nucleic acid probe supplied to the solid surface and the solid phase The maleimide group on the surface reacts to immobilize the nucleic acid probe. As a method for introducing a maleimide group, first, an aminosilane coupling agent is reacted with a glass substrate, and then the maleimide group is reacted by reacting the amino group with an EMCS reagent (N- (6-Maleimidocapropyloxy) succinimide: manufactured by Dojin). Introduce. Introduction of SH groups into DNA can be performed by using 5'-Thiol-Modifier C6 (manufactured by Glen Research) on an automatic DNA synthesizer. Examples of combinations of functional groups used for immobilization include combinations of epoxy groups (on the solid phase) and amino groups (ends of nucleic acid probes) in addition to the combinations of thiol groups and maleimide groups described above. . Further, surface treatment with various silane coupling agents is also effective, and oligonucleotides into which functional groups capable of reacting with functional groups introduced with the silane coupling agents are used. Furthermore, a method of coating a resin having a functional group can also be used.

  After performing the hybridization reaction, the surface of the 1205 DNA microarray is washed, the nucleic acid not bound to the probe is peeled off, (usually) dried, and the amount of fluorescence of 1206 is measured. Then, the substrate of the DNA microarray is irradiated with excitation light, and a fluorescent image is captured by the sensor (1207). The probe fluorescence intensity of the obtained DNA microarray fluorescence image (1208) is calculated.

  Next, the principle of a DNA microarray for identifying infectious bacteria will be described with reference to FIG. It is assumed that the DNA microarray shown in FIG. 13 is made for the purpose of specifying Staphylococcus aureus, for example.

  The left column is a treatment sequence derived from a wild strain of S. aureus, and the right column is a treatment sequence derived from a wild strain of Escherichia coli. For example, it can be considered that the flow on the left is a flow for processing blood of a patient infected with Staphylococcus aureus, and the flow on the right is a flow for processing blood of a patient infected with Escherichia coli.

  Both basically perform the same processing. That is, first, DNA is extracted from, for example, blood or sputum of a bacterially infected patient. In this case, generally, human DNA derived from somatic cells of a patient may be included.

  When the extracted DNA is small, amplification is performed by a method such as PCR. In this case, a fluorescent substance or a substance capable of binding the fluorescent substance is generally mixed as a label.

  When amplification is not performed, using the extracted DNA, a fluorescent substance or a substance capable of binding the fluorescent substance is mixed as a label while forming a complementary strand, or the directly extracted DNA is directly fluorescent or fluorescent. A substance capable of binding the substance is added as a label.

  Usually, when PCR amplification is performed, for the purpose of specifying the bacteria of an infectious disease, it is common to amplify a part of a base sequence constituting ribosomal RNA called so-called 16s rRNA. In this case, the PCR primer for S. aureus on the left and the PCR primer for E. coli on the right are almost the same. More specifically, multiplex PCR is performed using a primer set that can amplify the 16s rRNA coding portion of any fungus. The internal standard DNA prepared by the present invention is also amplified by multiplex PCR using the above primer set.

  If a DNA microarray designed for the purpose of determining S. aureus works correctly, the left hybrid solution will react positively with the spot and the right hybrid solution will react negatively with the spot.

  In exactly the same way, if a DNA microarray designed for the purpose of determining the presence of E. coli works correctly, the left hybrid solution reacts negatively and the right hybrid solution produces spotless Responds positively. Of course, infectious bacteria may be determined using a DNA microarray in which several types of spots that react specifically with various bacteria are arranged at the same time.

  The spot of the internal standard DNA detection probe reacts positively regardless of the presence of bacteria because the internal standard DNA is present. However, when the multiplex PCR fails for some reason, or when the hybridization reaction itself fails, it reacts negatively. This property allows PCR amplification assays to be performed. Furthermore, it is possible to determine how much luminance can be obtained by inserting the internal standard DNA from the amount of the internal standard DNA and the spot luminance of the probe for the internal standard DNA. By comparing the internal standard DNA with the sample, the quantitativeness of the sample can be improved.

(Embodiment 2)
Hereinafter, the operation flow described with reference to FIG. 13 will be described as a specific operation assuming the purpose of identifying the causative bacteria of the infectious disease. The organism type determination method is not limited to the identification of the causative bacteria of the infectious disease described below, and may be used for determination of human constitution such as MHC, and analysis of DNA and RNA related to diseases such as cancer.

<Preparation of target DNA probe>
Nucleic acid sequences (In) shown in Table 1 (n is a number) were designed as Probes for detecting Enterobacter cloacae.

  Specifically, the probe base sequence shown below was selected as a target DNA probe from the genome part coding 16s rRNA. These probe base sequence groups are designed to be highly specific to the bacteria and to be expected to have hybridization sensitivity that is sufficient and does not vary “as much as possible” with each probe base sequence.

  The target DNA probes shown in Table 1 were introduced with a thiol group at the 5 'end of the target DNA probe as a functional group for immobilization on the DNA microarray after synthesis according to a conventional method. After introduction of the functional group, it was purified and lyophilized. The freeze-dried probe for DNA of interest was stored in a freezer at −30 ° C.

  Staphylococcus aureus (An), Staphylococcus epidermidis (Bn), Escherichia coli (Cn), Neisseria pneumoniae (Dn), Pseudomonas aeruginosa (En), Serratia bacteria (Fn), The following table shows the target DNA probes for Streptococcus pneumoniae (G-n), Haemophilus influenzae (H-n), and Enterococcus faecalis (J-n) (n is a number). A probe set was designed.

<Preparation of probe for internal standard DNA>
The probe shown below was designed by the same method for the internal standard DNA probe.

<Preparation of PCR primer for target DNA>
Nucleic acid sequences shown in Table 12 were designed as primers for the target DNA for detection of pathogenic bacteria. Specifically, a primer set that specifically amplifies the genomic portion encoding Enterobacter cloacae 16s rRNA, that is, the specific melting temperature is aligned as much as possible at both ends of the approximately 1500 base length 16s rRNA coding region. Primers were designed. A plurality of types of primers were designed so that mutant strains and a plurality of 16s rRNA coding regions existing on the genome can be amplified simultaneously. If these primers are used, the internal standard DNA according to the present invention can also be amplified.

  The primers shown in Table 12 were synthesized and then purified by high performance liquid chromatography (HPLC), and 3 kinds of forward primer and 3 kinds of reverse primer were mixed, and each primer concentration was 10 pmol / μl final concentration. So that it was dissolved in TE buffer.

<Extraction of Enterobacter cloacae Genome DNA (model specimen)>
(Microbe culture & Genome DNA extraction pretreatment)
First, an Enterobacter cloacae standard strain was cultured according to a conventional method. 1.0 ml (OD600 = 0.7) of this microorganism culture solution was collected in a 1.5 ml capacity microtube, and the cells were collected by centrifugation (8500 rpm, 5 min, 4 ° C.). After discarding the supernatant, 300 μl of Enzyme Buffer (50 mM Tris-HCl: pH 8.0, 25 mM EDTA) was added and resuspended using a mixer. The resuspended bacterial solution was recovered again by centrifugation (8500 rpm, 5 min, 4 ° C.). After discarding the upper fine particles, the following enzyme solution was added to the collected cells and resuspended using a mixer.
Lysozyme 50 μl (20 mg / ml in Enzyme Buffer)
N-Acetylmuramidase SG 50 μl (0.2 mg / ml in Enzyme Buffer)

  Next, the bacterial solution resuspended by adding the enzyme solution was allowed to stand for 30 minutes in a 37 ° C. incubator to perform cell wall lysis treatment.

(Genome extraction)
Genome DNA extraction of the following microorganisms was performed using a nucleic acid purification kit (MagExtractor-Genome-: manufactured by TOYOBO).

  Specifically, first, 750 μl of the dissolution / adsorption solution and 40 μl of magnetic beads were added to the microorganism suspension obtained by dissolving the cell wall in the pretreatment, and vigorously stirred for 10 minutes using a tube mixer (step 1).

  Next, the microtube was set on a separation stand (Magnetic Trapper), allowed to stand for 30 seconds to collect magnetic particles on the wall surface of the tube, and the upper fine was discarded while being set on the stand (Step 2).

  Next, 900 μl of the washing solution was added, and the mixture was stirred for about 5 seconds with a mixer and re-suspended (step 3).

  Next, the microtube was set on a separation stand (Magnetic Trapper), allowed to stand for 30 seconds to collect magnetic particles on the wall surface of the tube, and the upper fine was discarded while being set on the stand (Step 4).

  Steps 3 and 4 were repeated to perform the second washing (Step 5), and then 900 μl of 70% ethanol was added and stirred for about 5 seconds with a mixer to re-suspend (Step 6).

  Next, the microtube was set on a separation stand (Magnetic Trapper), allowed to stand for 30 seconds to collect magnetic particles on the wall surface of the tube, and the upper fine was discarded while being set on the stand (Step 7).

  Steps 6 and 7 were repeated and the second washing with 70% ethanol (step 8) was performed. Then, 100 μl of pure water was added to the collected magnetic particles, and the mixture was stirred for 10 minutes with a tube mixer.

  Next, the microtube was set on a separation stand (Magnetic Trapper), allowed to stand for 30 seconds to collect magnetic particles on the tube wall surface, and the supernatant was collected in a new tube while being set on the stand.

(Inspection of recovered Genome DNA)
Genome DNA of the collected microorganism (Enterobacter cloacae strain) was subjected to agarose electrophoresis and absorbance measurement at 260/280 nm according to a conventional method, and the quality (contamination amount of low-molecular nucleic acid, degree of degradation) and the recovery amount were tested. .

  In this example, about 10 μg of Genome DNA was recovered, and Genome DNA degradation and rRNA contamination were not observed.

  The recovered Genome DNA was dissolved in TE buffer so as to have a final concentration of 50 ng / μl and used in the following examples.

<Production of DNA microarray>
[1] Cleaning of Glass Substrate A synthetic quartz glass substrate (size: 25 mm × 75 mm × 1 mm, manufactured by Iiyama Special Glass Co., Ltd.) was placed in a heat-resistant and alkali-resistant rack and immersed in a cleaning solution for ultrasonic cleaning adjusted to a predetermined concentration. After soaking in the cleaning solution overnight, ultrasonic cleaning was performed for 20 minutes. Subsequently, the substrate was taken out, rinsed lightly with pure water, and then ultrasonically cleaned in ultrapure water for 20 minutes. Next, the substrate was immersed in a 1N sodium hydroxide aqueous solution heated to 80 ° C. for 10 minutes. Pure water cleaning and ultrapure water cleaning were performed again to prepare a quartz glass substrate for a DNA chip.

[2] Surface treatment A silane coupling agent KBM-603 (manufactured by Shin-Etsu Silicone) was dissolved in pure water to a concentration of 1% and stirred at room temperature for 2 hours. Subsequently, the previously cleaned glass substrate was immersed in an aqueous silane coupling agent solution and allowed to stand at room temperature for 20 minutes. The glass substrate was pulled up and the surface was lightly washed with pure water, and then nitrogen gas was blown onto both sides of the substrate to dry it. Next, the dried substrate was baked in an oven heated to 120 ° C. for 1 hour to complete the coupling agent treatment, and amino groups were introduced onto the substrate surface. Next, N-maleimidocaproyloxy succinimide (N- (6-Maleimidocaproyloxy) succinimido) (hereinafter abbreviated as EMCS) manufactured by Dojindo Laboratories Co., Ltd. was dissolved in a 1: 1 mixed solvent of dimethyl sulfoxide and ethanol. An EMCS solution dissolved to a concentration of 0.3 mg / ml was prepared. The glass substrate after baking was allowed to cool and immersed in the prepared EMCS solution for 2 hours at room temperature. By this treatment, the amino group introduced on the surface by the silane coupling agent and the succinimide group of EMCS reacted to introduce a maleimide group on the glass substrate surface. The glass substrate pulled up from the EMCS solution was washed with the above-mentioned mixed solvent in which MCS was dissolved, further washed with ethanol, and then dried in a nitrogen gas atmosphere.

[3] Probe DNA
The prepared target DNA probe and internal standard DNA probe were dissolved in pure water, dispensed to a final concentration (at the time of ink dissolution) of 10 μM, and then freeze-dried to remove moisture.

[4] DNA ejection by BJ printer and binding to substrate Aqueous solution containing 7.5 wt% glycerin, 7.5 wt% thiodiglycol, 7.5 wt% urea, 1.0 wt% acetylenol EH (manufactured by Kawaken Fine Chemical Co., Ltd.) Prepared. Subsequently, a total of 61 types of probes, ie, 60 types of the target DNA probes (Tables 1 to 10) and one type of internal standard DNA probe were dissolved in the above mixed solvent so as to have a prescribed concentration. The obtained DNA probe solution was filled in an ink tank for a bubble jet printer (trade name: BJF-850 manufactured by Canon Inc.) and mounted on a print head.

  The bubble jet printer used here is modified so that printing on a flat plate is possible. The bubble jet printer can spot a DNA solution of about 5 picoliters at a pitch of about 120 micrometers by inputting a print pattern according to a predetermined file creation method.

  Subsequently, using this modified bubble jet printer, a printing operation was performed on one glass substrate to produce an array. After confirming that printing was performed reliably, it was left in a humidified chamber for 30 minutes to react the maleimide group on the surface of the glass substrate with the thiol group at the end of the probe.

[5] Washing After the reaction for 30 minutes, the DNA solution remaining on the surface was washed away with 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl, and the probe (single-stranded DNA) was immobilized on the glass substrate surface. A DNA microarray was obtained.

<Amplification and labeling of specimen (PCR amplification & incorporation of fluorescent label)>
The amplification and labeling reaction of the target DNA of the microorganism serving as the specimen are shown below. Template Genome DNA is Genome DNA of Enterobacter cloacae strain that has been modified by the previous operation. “Forward Primer mix” is a primer solution containing three kinds of forward primers shown in Table 12.
Premix PCR reagent (TAKARA ExTaq) 25μl
Template Genome DNA 2μl (100ng)
Internal standard DNA 2μl (100ng)
Forward Primer mix 2μl (20 pmol/tube each)
Reverse Primer mix 2μl (20 pmol/tube each)
Cy-3 dUTP (1mM) 2μl (2nmol / tube)
H ₂ 0 15 μl
Total 50μl

The reaction solution having the above composition was subjected to an amplification reaction using a commercially available thermal cycler according to the following protocol.
(1) 95 ℃ 10 min.
(2) 92 ° C 45 sec.
(3) 55 ° C 45 sec.
(4) 72 ° C 45 sec.
(5) 72 ° C 10 min.
(2) to (4) were performed 35 cycles.
After completion of the reaction, the primer was removed using a purification column (QIAGEN QIAquick PCR Purification Kit), and the amplification product was quantified to obtain a labeled sample.

<Hybridization>
Detection reaction was performed using the DNA microarray prepared in <Preparation of DNA microarray> and the labeled sample prepared in <Amplification and labeling of specimen (PCR amplification & incorporation of fluorescent label)>.
(Blocking of DNA microarray)
BSA (bovine serum albumin Fraction V: manufactured by Sigma) was dissolved in 100 mM NaCl / 10 mM Phosphate Buffer so as to be 1 wt%, and the DNA microarray prepared in <Preparation of DNA microarray> was immersed in this solution at room temperature for 2 hours. Blocking was performed. After completion of blocking, 2 × SSC solution containing 0.1 wt% SDS (sodium dodecyl sulfate) (NaCl 300 mM, sodium citrate (Trisodium citrate dihydrate, C ₆ H ₅ Na ₃ .2H ₂ O) 30 mM, pH 7.0) After washing with, rinsed with pure water and drained with a spin dryer.

(Hybridization)
The drained DNA microarray was set in a hybridization apparatus (Genomic Solutions Inc. Hybridization Station), and a hybridization reaction was carried out under the following hybridization solution and conditions.

<Hybridization solution>
6 x SSPE / 10% Form amide / Target (PCR Products total amount)
_{(6xSSPE: NaCl 900mM, NaH 2} PO 4 · H 2 O 60mM, EDTA 6mM, pH 7.4)

<Hybridization conditions>
65 ° C 3 min → 92 ° C 2 min → 45 ° C 3 hr → Wash 2 x SSC / 0.1% SDS at 25 ° C → Wash 2 x SSC at 20 ° C → (Rinse with H ₂ O: Manual) → Spin dry

<Detection of microorganisms (fluorescence measurement)>
The DNA microarray after completion of the hybridization reaction was subjected to fluorescent image capturing using the apparatus of FIG. FIG. 11 shows a fluorescent image derived from Enterobacter cloacae obtained as a result. Here, the presence / absence of Enterobacter cloacae is determined by digitizing the fluorescence brightness of the probe spot.

Schematic showing the positional relationship between DNA encoding 16s rRNA in DNA and target DNA amplified by the target DNA primer Schematic showing the positional relationship between the target DNA and the target DNA primer. Schematic showing the target DNA used as the amplification object of PCR reaction. Schematic showing internal standard DNA produced by the manufacturing method which concerns on this invention. It is the figure which showed the flow of the internal standard DNA manufacturing method of this invention. It is a figure for demonstrating the outline | summary of the sequence replacement method of the internal standard candidate DNA of this invention. It is the figure which showed alignment with the forward / reverse direction primer of internal standard candidate DNA. It is a figure for demonstrating the example of a sequence substitution of internal standard candidate DNA. It is a block diagram which shows the structure of the information processing apparatus for implement | achieving this invention. It is the figure which showed the structure of the fluorescence image imaging device of a DNA microarray. It is the figure which showed the mode of the hybridization on a DNA microarray. It is a figure for demonstrating the whole experimental procedure of the hybridization reaction experiment using a DNA microarray. It is a figure for demonstrating the principle of the DNA microarray which identifies the microbe of an infectious disease. It is the figure which showed the DNA microarray fluorescence image. It is the figure which showed the arrangement | sequence of the internal standard DNA produced by this invention.

Explanation of symbols

11 DNA encoding 16S rRNA
12 DNA of interest
13 DNA
21 Forward primer (primer for target DNA)
22 Reverse primer (primer for target DNA)
31 5 ′ end region of target DNA 32 central region of target DNA 33 3 ′ end region of target DNA 41 5 ′ end region of internal standard DNA 42 central region of internal standard DNA 43 3 ′ end region of internal standard DNA 44 Forward direction Primer (primer for target DNA)
45 Reverse primer (primer for target DNA)

Claims (2)

A method for producing an internal standard DNA that is added to a reaction solution of a PCR reaction for amplifying a target DNA derived from a detection target bacterium and amplified by a target DNA primer for amplifying the target DNA,
Select internal standard candidate DNA satisfying the following conditions by database search,
The sequences of the 5 ′ terminal region and 3 ′ terminal region of the internal standard candidate DNA are the same as the complementary sequences of the forward primer sequence and the reverse primer of the target DNA primer, respectively, by PCR using a replacement primer. A method for producing an internal standard DNA, characterized in that
Condition (A): a sequence partially including a region having a predetermined homology with the forward primer (hereinafter abbreviated as region of Condition A).
Condition (B): a sequence partially including a region having a predetermined homology with a complementary sequence of the reverse primer (hereinafter abbreviated as a region of Condition B).
Condition (C): a sequence in which the region of Condition A is on the 5 ′ end side and the region of Condition B is on the 3 ′ end side.
Condition (D): The sequence between the region of the condition A and the region of the condition B has the same number of bases as the target DNA, and has low homology with the 16S rRNA sequence of the detection target bacteria. The method for producing an internal standard DNA according to claim 1, wherein the internal standard candidate DNA further satisfies the condition (E).
Condition (E): a sequence in which no sequence having homology with the target DNA probe hybridizing with the target DNA exists between the region of the condition A and the region of the condition B.